Compositions and methods for non-parenteral delivery of oligonucleotides

ABSTRACT

The present invention relates to compositions and methods which enhance the local and systemic uptake and delivery of oligonucleotides and nucleic acids via non-parenteral routes of administration. Pharmaceutical compositions comprising oligonucleotides disclosed herein include, for systemic delivery, emulsion and microemulsion formulations for a variety of applications and oral dosage formulations. It has also surprisingly been discovered that oligonucleotides may be locally delivered to colonic sites by rectal enemas and suppositories in simple solutions, e.g., neat or in saline. Such pharmaceutical compositions of oligonucleotides may further include one or more penetration enhancers for the transport of oligonucleotides and other nucleic acids across mucosal membranes. The compositions and methods of the invention are utilized to effect the oral, buccal, rectal or vaginal administration of an antisense oligonucleotide to an animal in order to modulate the expression of a gene in the animal for investigative, therapeutic, palliative or prophylactic purposes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application continuation of U.S. patent application Ser. No.11/237,063, filed Sep. 28, 2005, now U.S. Pat. No. 8,377,897, which is acontinuation of U.S. patent application Ser. No. 09/315,298 filed May20, 1999, now abandoned, which is a continuation-in-part of U.S. patentapplication Ser. No. 09/108,673 filed Jul. 1, 1998, now U.S. Pat. No.6,887,906, which is a continuation-in-part of U.S. patent applicationSer. No. 08/886,829, filed Jul. 1, 1997, now abandoned, the disclosuresof which are incorporated by reference herein in their entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledISIS3510USC2SEQ.txt, created on Jan. 4, 2013 which is 12 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compositions and methods which enhancethe local and systemic uptake and delivery of nucleic acids vianon-parenteral routes of administration. More particularly, the methodsand compositions enhance the transport of oligonucleotides and othernucleic acids across mucosal membranes through the use of one or morepenetration enhancers. The compositions of the present invention aresolutions, emulsions, and related mixtures that facilitate the uptakeand delivery of oligonucleotides and other nucleic acids. The presentinvention is directed to the use of various fatty acids, bile salts,chelating agents and other penetration enhancers, as well as carriercompounds, to enhance the stability of oligonucleotides and othernucleic acids and/or their transport across cell walls and/or intocells. More specific objectives and advantages of the invention willhereinafter be made clear or become apparent to those skilled in the artduring the course of explanation of preferred embodiments of theinvention.

BACKGROUND OF THE INVENTION

Advances in the field of biotechnology have led to significant advancesin the treatment of diseases such as cancer, genetic diseases, arthritisand AIDS that were previously difficult to treat. Many such advancesinvolve the administration of oligonucleotides and other nucleic acidsto a subject, particularly a human subject. The administration of suchmolecules via parenteral routes has been shown to be effective for thetreatment of diseases and/or disorders. See, e.g., Draper et al., U.S.Pat. No. 5,595,978, Jan. 21, 1997, which discloses intravitrealinjection as a means for the direct delivery of antisenseoligonucleotides to the vitreous humor of the mammalian eye. See also,Robertson, Nature Biotechnology, 1997, 15, 209, and Genetic EngineeringNews, 1997, 15, 1, each of which discuss the treatment of Crohn'sdisease via intravenous infusions of antisense oligonucleotides.Non-parenteral routes for administration of oligonucleotides and othernucleic acids (such as oral or rectal delivery or other mucosal routes)offers the promise of simpler, easier and less injurious administrationof such nucleic acids without the need for sterile procedures and theirconcomitant expenses, e.g., hospitalization and/or physician fees. Therethus is a need to provide compositions and methods to enhance theavailability of novel drugs such as oligonucleotides when administeredvia non-parenteral routes. It is desirable that such new compositionsand methods provide for the simple, convenient, practical and optimalnon-parenteral delivery of oligonucleotides and other nucleic acids.

SUMMARY OF THE INVENTION

In accordance with the present invention, compositions and methods areprovided for the non-parenteral delivery and mucosal penetration ofnucleic acids in an animal. In particular, the present inventionprovides compositions and methods for modulating the production ofselected proteins or other biological phenomena in an animal, whichinvolves the administration of an oligonucleotide, especially anantisense oligonucleotide, via non-parenteral means to an animal,thereby circumventing the complications and expense which may beassociated with intravenous and other parenteral modes of in vivoadministration. “Non-parenteral administration” refers to thecontacting, directly or otherwise, to all or a portion of the alimentarycanal, skin, eyes, pulmonary tract, urethra or vagina of an animal.Compositions of the present invention may be a mixture of components orphases as are present in emulsions (including microemulsions andcreams), and related formulations comprising two or more phases.

In one aspect, the present invention provides pharmaceuticalcompositions comprising at least one nucleosidic moiety such as anucleoside, nucleotide, or nucleic acid in a solution or emulsion. Thenucleic acid can be a ribozyme, a PNA, or an aptamer, but preferably isan oligonucleotide such as, for example, an oligonucleotide thatmodulates expression of a cellular adhesion protein, modulates a rate ofcellular proliferation, or has biological activity against eukaryoticpathogens or retroviruses.

In certain embodiments, solutions according to the invention consistessentially of the nucleosidic moiety and a solvent comprising, forexample, saline solution or cocoa butter. Emulsions according to theinvention include oil-in-water emulsions, water-in-oil emulsions,oil-in-water-in-oil emulsions, and water-in-oil-in-water emulsions.

In certain embodiments, the pharmaceutical compositions of the inventionfurther comprise a penetration enhancer such as a fatty acid, a bilesalt, a chelating agent, a surfactant, and a non-surfactant such as anunsaturated cyclic urea, a 1-alkyl-alkanone, a1-alkenylazacyclo-alakanone, or a steroidal anti-inflammatory agent.

Also provided are methods for treating an animal comprisingadministering to the animal a therapeutically effective amount of apharmaceutical composition according to the invention. The compositioncan be administered by, for example, buccal, sublingual, endoscopic,rectal, oral, vaginal, topical, pulmonary, or urethral routes. Inpreferred embodiments, the compositions of the invention areadministered rectally means of an enema or a suppository.

Because of the advantages of non-parenteral delivery of drugs of theantisense class, the compositions and methods of the invention can beused in therapeutic methods as explained in more detail herein. Thecompositions and methods herein provided may also be used to examine thefunction of various proteins and genes in an animal, including thoseessential to animal development. The methods of the invention can beused, for example, for the treatments of animals that are known orsuspected to suffer from diseases such as ulcerative colitis, Chrohn'sdisease, inflammatory bowel disease, or undue cellular proliferation.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions and methods for the local as well assystemic delivery of oligonucleotides and other nucleic acids to ananimal via non-parenteral means. In particular, the present inventionprovides compositions and methods for modulating the in vivo expressionof a gene in an animal through the non-parenteral administration of anantisense oligonucleotide, thereby circumventing the complications andexpense which may be associated with intravenous and other parenteralroutes of administration.

Enhanced bioavailability of oligonucleotides and other nucleic acids isachieved via the non-parenteral administration of the compositions andmethods of the present invention. The term “bioavailability” refers to ameasurement of what portion of an administered drug reaches thecirculatory system when a non-parenteral mode of administration is usedto introduce the drug into an animal. The term is used for drugs whoseefficacy is related to the blood concentration achieved, even if thedrug's ultimate site of action is intracellular (van Berge-Henegouwen etal., Gastroenterol., 1977, 73, 300). Traditionally, bioavailabilitystudies determine the degree of intestinal absorption of a drug bymeasuring the change in peripheral blood levels of the drug after anoral dose (DiSanto, Chapter 76 In: Remington's Pharmaceutical Sciences,18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages1451-1458). The area under the curve (AUC₀) is divided by the area underthe curve after an intravenous (i.v.) dose (AUC_(iv)) and the quotientis used to calculate the fraction of drug absorbed. This approach cannotbe used, however, with compounds which have a large “first passclearance,” i.e., compounds for which hepatic uptake is so rapid thatonly a fraction of the absorbed material enters the peripheral blood.For such compounds, other methods must be used to determine the absolutebioavailability (van Berge-Henegouwen et al., Gastroenterol., 1977, 73,300). With regards to oligonucleotides, studies suggest that they arerapidly eliminated from plasma and accumulate mainly in the liver andkidney after i.v. administration (Miyao et al., Antisense Res. Dev.,1995, 5, 115; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996,6, 177).

Another “first pass effect” that applies to orally administered drugs isdegradation due to the action of gastric acid and various digestiveenzymes. Furthermore, the entry of many high molecular weight activeagents (such as peptides, proteins and oligonucleotides) and someconventional and/or low molecular weight drugs (e.g., insulin,vasopressin, leucine enkephalin, etc.) through mucosal routes (such asoral, pulmonary, buccal, rectal, transdermal, vaginal and ocular) to thebloodstream is frequently obstructed by poor transport across epithelialcells and concurrent metabolism during transport. This type ofdegradative metabolism is known for oligonucleotides and nucleic acids.For example, phosphodiesterases are known to cleave the phosphodiesterlinkages of oligonucleotides and many other modified linkages present insynthetic oligonucleotides and nucleic acids.

One means of ameliorating first pass clearance effects is to increasethe dose of administered drug, thereby compensating for proportion ofdrug lost to first pass clearance. Although this may be readily achievedwith i.v. administration by, for example, simply providing more of thedrug to an animal, other factors influence the bioavailability of drugsadministered via non-parenteral means. For example, a drug may beenzymatically or chemically degraded in the alimentary canal or bloodstream and/or may be impermeable or semipermeable to various mucosalmembranes.

It has now been found that oligonucleotides can be introducedeffectively into animals via non-parenteral means throughcoadministration of “mucosal penetration enhancers,” also known as“absorption enhancers” or simply as “penetration enhancers”. These aresubstances which facilitate the transport of a drug across mucousmembrane(s) associated with the desired mode of administration.

A “pharmaceutically acceptable” component of a formulation of theinvention is one which, when used together with excipients, diluents,stabilizers, preservatives and other ingredients are appropriate to thenature, composition and mode of administration of a formulation.Accordingly it is desired to select penetration enhancers whichfacilitate the uptake of oligonucleotides, without interfering with theactivity of the oligonucleotides and in a manner such that the same canbe introduced into the body of an animal without unacceptableside-effects such as toxicity, irritation or allergic response.

The present invention provides compositions comprising one or morepharmaceutically acceptable penetration enhancers, and methods of usingsuch compositions, which result in the improved bioavailability ofnucleic acids administered via non-parenteral modes of administration.Heretofore, certain penetration enhancers have been used to improve thebioavailability of certain drugs. See Muranishi, Crit. Rev. Ther. DrugCarrier Systems, 1990, 7, 1 and Lee et al., Crit. Rev. Ther. DrugCarrier Systems, 1991, 8, 91. However, it is generally viewed to be thecase that effectiveness of such penetration enhancers is unpredictable.Therefore, it has been surprisingly found that the uptake and deliveryof oligonucleotides, relatively complex molecules which are known to bedifficult to administer to animals and man, can be greatly improved evenwhen administered by non-parenteral means through the use of a number ofdifferent classes of penetration enhancers.

The effective non-parenteral use and administration of compositions ofthe present invention involves consideration of a number of differentaspects about drug therapy. One important consideration when using thecompositions and methods of the present invention is the mode ofadministration of the pharmaceutical composition containing thetherapeutic oligonucleotide or other nucleic acid. Administrationtypically is either parenteral or non-parenteral. Non-parenteral modesof administration include, but are not limited to, buccal, sublingual,endoscopic, oral, rectal, transdermal, topical, nasal, intratracheal,pulmonary, urethral, vaginal, and ocular. When administered by suchnon-parenteral modes the methods and composition of the presentinvention can deliver drug both locally and systemically as desired.

A second consideration of importance when using the compositions andmethods of the present invention is the use and nature of penetrationenhancers and carriers. Penetration enhancers facilitate the transportof drug molecules, for example, oligonucleotides and other nucleicacids, across mucosal and other epithelial cell membranes. Penetrationenhancers include, but are not limited to, members of molecular classessuch as surfactants, fatty acids, bile salts, chelating agents, andnon-chelating non-surfactant molecules. Carriers are inert moleculesthat may be included in the compositions of the present invention tointerfere with processes that lead to reduction in the levels ofbioavailable nucleic acid or oligonucleotide drug.

A third consideration of importance to the compositions and methods ofthe present invention is the nature of oligonucleotide or other nucleicacid used. Oligonucleotides of the present invention may be, but are notlimited to, those nucleic acids bearing modified linkages, modifiednucleobases, or modified sugars, and chimeric nucleic acids.

A fourth consideration of importance in the present invention is thenature of the composition. Pharmaceutical compositions of the presentinvention include, but are not limited to, solutions, emulsions(including microemulsions and creams), and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Thecompositions of the present invention may be formulated into any of manypossible dosage forms such as, but not limited to, tablets, capsules,liquid syrups, soft gels, suppositories, and enemas.

A fifth consideration of importance to the compositions and methods ofthe present invention is the means by which such compositions may beadministered. Thus the dose, method of administration or application,and the use of additives are all worthy of consideration in this regard.Further, the methods and compositions of the present invention may beused to ameliorate a variety of diseases via local or systemictreatment. Such local or systemic treatment may be accomplished usingthe methods and compositions of the present invention via modes ofadministration that include, but are not limited to, buccal, sublingual,endoscopic, oral, rectal, transdermal, topical, nasal, pulmonary,urethral, vaginal, and ocular modes.

A sixth consideration of importance to the compositions and methods ofthe present invention is their applicability to bioequivalents ofoligonucleotides and other nucleic acids such as, but not limited to,oligonucleotide prodrugs, deletion derivatives, conjugates, aptamers,and ribozymes.

The present invention provides compositions and methods for local andsystemic delivery of one or more nucleic acids to an animal vianon-parenteral administration. For purposes of the invention, the term“animal” is meant to encompass humans as well as other mammals, as wellas reptiles, fish, amphibians, and birds. The term “non-parenteraldelivery” refers to the administration, directly or otherwise, of thedrug via a non-invasive procedure which typically does not entail theuse of a syringe and needle. Non-parenteral administration may be, butis not limited to, delivery of the drug via the alimentary canal or viatransdermal, topical, nasal, pulmonary, urethral, vaginal or ocularroutes. The term “alimentary canal” refers to the tubular passage in ananimal that functions in the digestion and absorption of food and theelimination of food residue, which runs from the mouth to the anus, andany and all of its portions or segments, e.g., the oral cavity, theesophagus, the stomach, the small and large intestines and the colon, aswell as compound portions thereof such as, e.g., the gastro-intestinaltract. Thus, the term “alimentary delivery” encompasses several routesof administration including, but not limited to, oral, rectal,endoscopic and sublingual/buccal administration. A common requirementfor these modes of administration is absorption over some portion or allof the alimentary tract and a need for efficient mucosal penetration ofthe nucleic acid(s) so administered.

In addition, iontophoresis (transfer of ionic solutes through biologicalmembranes under the influence of an electric field) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 163),phonophoresis or sonophoresis (use of ultrasound to enhance theabsorption of various therapeutic agents across biological membranes,notably the skin and the cornea) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 166), and optimization ofvehicle characteristics relative to dose deposition and retention at thesite of administration (Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 168) may be useful methods for enhancing thetransport of drugs across mucosal sites in accordance with the presentinvention.

Delivery of a drug via the oral mucosa, as in the case of buccal andsublingual administration, has several desirable features, including, inmany instances, a more rapid rise in plasma concentration of the drugthan via oral delivery (Harvey, Chapter 35 In: Remington'sPharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co.,Easton, Pa., 1990, page 711). Furthermore, because venous drainage fromthe mouth is to the superior vena cava, this route also bypasses rapidfirst-pass metabolism by the liver. Both of these features contribute tothe sublingual route being the mode of choice for drugs likenitroglycerin (Benet et al., Chapter 1 In: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds.,McGraw-Hill, New York, N.Y., 1996, page 7).

Endoscopy may be used for drug delivery directly to an interior portionof the alimentary tract. For example, endoscopic retrogradecystopancreatography (ERCP) takes advantage of extended gastroscopy andpermits selective access to the biliary tract and the pancreatic duct(Hirahata et al., Gan To Kagaku Ryoho, 1992, 19(10 Suppl.), 1591).Pharmaceutical compositions, including liposomal formulations, can bedelivered directly into portions of the alimentary canal, such as, e.g.,the duodenum (Somogyi et al., Pharm. Res., 1995, 12, 149) or the gastricsubmucosa (Akamo et al., Japanese J. Cancer Res., 1994, 85, 652) viaendoscopic means. Gastric lavage devices (Inoue et al., Artif. Organs,1997, 21, 28) and percutaneous endoscopic feeding devices (Pennington etal., Ailment Pharmacol. Ther., 1995, 9, 471) can also be used for directalimentary delivery of pharmaceutical compositions.

Drugs administered by the oral route can often be alternativelyadministered by the lower enteral route, i.e., through the anus into therectum or lower intestine. Rectal suppositories, retention enemas orrectal catheters can be used for this purpose and may be preferred whenpatient compliance might otherwise be difficult to achieve (e.g., inpediatric and geriatric applications, or when the patient is vomiting orunconscious). Rectal administration can result in more prompt and higherblood levels than the oral route. (Harvey, Chapter 35 In: Remington'sPharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co.,Easton, Pa., 1990, page 711). Because about 50% of the drug that isabsorbed from the rectum will bypass the liver, administration by thisroute significantly reduces the potential for first-pass metabolism(Benet et al., Chapter 1 In: Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, NewYork, N.Y., 1996).

The preferred method of non-parenteral administration for most drugs isoral delivery. This is typically the most convenient route for access tothe systemic circulation. Absorption from the alimentary canal isgoverned by factors that are generally applicable, e.g., surface areafor absorption, blood flow to the site of absorption, the physical stateof the drug and its concentration at the site of absorption (Benet etal., Chapter 1 In: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York,N.Y., 1996, pages 5-7). A significant factor which may limit the oralbioavailability of a drug is the degree of “first pass effects.” Forexample, some substances have such a rapid hepatic uptake that only afraction of the material absorbed enters the peripheral blood (VanBerge-Henegouwen et al., Gastroenterology, 1977, 73:300). Thecompositions and methods of the invention circumvent, at leastpartially, such first pass effects by providing improved uptake ofnucleic acids by, e.g., causing the hepatic uptake system to becomesaturated and allowing a significant portion of the nucleic acid soadministered to reach the peripheral circulation.

Topical administration is often chosen when local delivery of a drug isdesired at, or immediately adjacent to the point of application of thedrug composition or formulation. Although occasionally enough drug isabsorbed into the systemic circulation to cause systemic effects,topical routes generally are not effective for systemic therapy. Threegeneral types of topical routes of administration are recognized,topical administration of a drug composition to mucous membranes, skinor eyes.

Drugs that are applied to the mucous membranes produce primarily localeffects. This route of administration includes application of drugcompositions to mucous membranes of the conjunctiva, nasopharynx,oropharynx, vagina, colon, urethra, and urinary bladder. Absorption ofdrugs occurs rapidly through mucous membranes and is an effective routefor localized therapy and, on occasion, for systemic therapy.

Transdermal drug delivery is a valuable route for the administration oflipid soluble therapeutics. It has been recognized that the dermis ismore permeable than the epidermis and therefore absorption of drugs ismuch more rapid through abraded, burned or denuded skin. Inflammationand other physiologic conditions that increase blood flow to the skinalso enhance absorption via the transdermal route. Absorption by thisroute may be enhanced via the use of an oily vehicle (inunction) orthrough the use of penetration enhancers. Hydration of the skin and theuse of controlled release topical patches are also effective ways toadminister drugs via the transdermal route. This route provides a meansto deliver the drug for both systemic and local therapy.

Ocular delivery of drugs is especially useful for the local treatment ofeye infections or abnormalities. The drug is typically administered viainstillation and absorption of the drug occurs through the cornea.Corneal infection or trauma may thus result in more rapid absorption.Ophthalmic delivery systems that provide prolonged duration of action(e.g., suspensions and ointments) and ocular inserts that providecontinuous delivery of low amounts of drugs are useful additions toophthalmic therapy. The ocular delivery of drugs results inpredominantly local effects. Systemic absorption that results fromdrainage via the nasolachrimal canal is limited and few systemic sideeffects are typically observed.

The present invention employs various penetration enhancers in order toeffect transport of oligonucleotides and other nucleic acids acrossmucosal and epithelial membranes. Penetration enhancers may beclassified as belonging to one of five broad categories—surfactants,fatty acids, bile salts, chelating agents, and non-chelatingnon-surfactants (Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of these classes is discussed inmore detail in the following paragraphs. Carrier substances (or simply“carriers”), which reduce first pass effects by, e.g., saturating thehepatic uptake system, are also herein described.

In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of oligonucleotides through thealimentary mucosa and other epithelial membranes is enhanced. Inaddition to bile salts and fatty acids, surfactants include, forexample, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether andpolyoxyethylene-20-cetyl ether (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and perfluorochemicalemulsions, such as FC-43 (Takahashi et al., J. Pharm. Phamacol., 1988,40, 252).

Fatty acids and their derivatives which act as penetration enhancers andmay be used in compositions of the present invention include, forexample, oleic acid, lauric acid, capric acid (n-decanoic acid),myristic acid, palmitic acid, stearic acid, linoleic acid, linolenicacid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol),dilaurin, caprylic acid, arachidonic acid, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines and mono-and di-glycerides thereof and/or physiologically acceptable saltsthereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate,linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1; El-Hariri et al., J.Pharm. Pharmacol., 1992, 44, 651).

A variety of bile salts also function as penetration enhancers tofacilitate the uptake and bioavailability of drugs. The physiologicalroles of bile include the facilitation of dispersion and absorption oflipids and fat-soluble vitamins (Brunton, Chapter 38 In: Goodman &Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman etal., eds., McGraw-Hill, New York, N.Y., 1996, pages 934-935). Variousnatural bile salts, and their synthetic derivatives, act as penetrationenhancers. Thus, the term “bile salt” includes any of the naturallyoccurring components of bile as well as any of their syntheticderivatives. The bile salts of the invention include, for example,cholic acid (or its pharmaceutically acceptable sodium salt, sodiumcholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid(sodium deoxycholate), glucholic acid (sodium glucholate), glycholicacid (sodium glycocholate), glycodeoxycholic acid (sodiumglycodeoxycholate), taurocholic acid (sodium taurocholate),taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid(CDCA, sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodiumtauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate andpolyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579).

In a particular embodiment, penetration enhancers useful in the presentinvention are mixtures of penetration enhancing compounds. For example,a particularly preferred penetration enhancer is a mixture of UDCA(and/or CDCA) with capric and/or lauric acids or salts thereof e.g.sodium. Such mixtures are useful for enhancing the delivery ofbiologically active substances across mucosal membranes, in particularintestinal mucosa. Preferred penetration enhancer mixtures compriseabout 5-95% of bile acid or salt(s) UDCA and/or CDCA with 5-95% capricand/or lauric acid. Particularly preferred are mixtures of the sodiumsalts of UDCA, capric acid and lauric acid in a ratio of about 1:2:2respectively. In another particularly preferred embodiment

Chelating agents, as used in connection with the present invention, canbe defined to be compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption ofoligonucleotides through the alimentary and other mucosa is enhanced.With regards to their use as penetration enhancers in the presentinvention, chelating agents have the added advantage of also serving asDNase inhibitors, as most characterized DNA nucleases require a divalentmetal ion for catalysis and are thus inhibited by chelating agents(Jarrett, J. Chromatogr., 1993, 618, 315). Chelating agents of theinvention include, but are not limited to, disodiumethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1; Buur et al., J ControlRel., 1990, 14, 43).

As used herein, non-chelating non-surfactant penetration enhancers maybe defined as compounds that demonstrate insignificant activity aschelating agents or as surfactants but that nonetheless enhanceabsorption of oligonucleotides through the alimentary and other mucosalmembranes (Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1). This class of penetration enhancers includes, butis not limited to, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621).

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), can be used.

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate oligonucleotide in hepatic tissue can be reduced whenit is coadministered with polyinosinic acid, dextran sulfate,polycytidic acid or 4-acetamido-4′ isothiocyano-stilbene-2,2′-disulfonicacid (Miyao et al., Antisense Res. Dev., 1995, 5, 115; Takakura et al.,Antisense & Nucl. Acid Drug Dev., 1996, 6, 177).

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinised maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, EXPLOTAB); and wetting agents (e.g., sodium laurylsulphate, etc.).

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecomposition of present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention.

The present invention employs oligonucleotides for use in antisensemodulation of the function of DNA or messenger RNA (mRNA) encoding aprotein the modulation of which is desired, and ultimately to regulatethe amount of such a protein. Hybridization of an antisenseoligonucleotide with its mRNA target interferes with the normal role ofmRNA and causes a modulation of its function in cells. The functions ofmRNA to be interfered with include all vital functions such astranslocation of the RNA to the site for protein translation, actualtranslation of protein from the RNA, splicing of the RNA to yield one ormore mRNA species, turnover or degradation of the mRNA and possibly evenindependent catalytic activity which may be engaged in by the RNA. Theoverall effect of such interference with mRNA function is modulation ofthe expression of a protein, wherein “modulation” means either anincrease (stimulation) or a decrease (inhibition) in the expression ofthe protein. In the context of the present invention, inhibition is thepreferred form of modulation of gene expression.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid or deoxyribonucleic acid.This term includes oligonucleotides composed of naturally-occurringnucleobases, sugars and covalent intersugar (backbone) linkages as wellas modified oligonucleotides having non-naturally-occurring portionswhich function similarly. Such modified or substituted oligonucleotidesare often preferred over native forms because of desirable propertiessuch as, for example, enhanced cellular uptake, enhanced binding totarget and increased stability in the presence of nucleases.

An oligonucleotide is a polymer of repeating units generically known asa nucleotides. An unmodified (naturally occurring) nucleotide has threecomponents: (1) a nitrogenous base linked by one of its nitrogen atomsto (2) a 5-carbon cyclic sugar and (3) a phosphate, esterified to carbon5 of the sugar. When incorporated into an oligonucleotide chain, thephosphate of a first nucleotide is also esterified to carbon 3 of thesugar of a second, adjacent nucleotide. The “backbone” of an unmodifiedoligonucleotide consists of (2) and (3), that is, sugars linked togetherby phosphodiester linkages between the carbon 5 (5′) position of thesugar of a first nucleotide and the carbon 3 (3′) position of a second,adjacent nucleotide. A “nucleoside” is the combination of (1) anucleobase and (2) a sugar in the absence of (3) a phosphate moiety(Kornberg, A., DNA Replication, W.H. Freeman & Co., San Francisco, 1980,pages 4-7). The backbone of an oligonucleotide positions a series ofbases in a specific order; the written representation of this series ofbases, which is conventionally written in 5′ to 3′ order, is known as anucleotide sequence.

Oligonucleotides may comprise nucleotide sequences sufficient inidentity and number to effect specific hybridization with a particularnucleic acid. Such oligonucleotides which specifically hybridize to aportion of the sense strand of a gene are commonly described as“antisense.” In the context of the invention, “hybridization” meanshydrogen bonding, which may be Watson-Crick, Hoogsteen or reversedHoogsteen hydrogen bonding, between complementary nucleotides. Forexample, adenine and thymine are complementary nucleobases which pairthrough the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat an oligonucleotide need not be 100% complementary to its target DNAsequence to be specifically hybridizable. An oligonucleotide isspecifically hybridizable when binding of the oligonucleotide to thetarget DNA or RNA molecule interferes with the normal function of thetarget DNA or RNA to cause a decrease or loss of function, and there isa sufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment, or in the case of invitro assays, under conditions in which the assays are performed.

Antisense oligonucleotides are commonly used as research reagents,diagnostic aids, and therapeutic agents. For example, antisenseoligonucleotides, which are able to inhibit gene expression withexquisite specificity, are often used by those of ordinary skill toelucidate the function of particular genes, for example to distinguishbetween the functions of various members of a biological pathway. Thisspecific inhibitory effect has, therefore, been harnessed by thoseskilled in the art for research uses. Antisense oligonucleotides havealso been used as diagnostic aids based on their specific binding orhybridization to DNA or mRNA that are present in certain disease statesand due to the high degree of sensitivity that hybridization basedassays and amplified assays that utilize some of polymerase chainreaction afford. The specificity and sensitivity of oligonucleotides isalso harnessed by those of skill in the art for therapeutic uses. Forexample, the following U.S. patents demonstrate palliative, therapeuticand other methods utilizing antisense oligonucleotides. U.S. Pat. No.5,135,917 provides antisense oligonucleotides that inhibit humaninterleukin-1 receptor expression. U.S. Pat. No. 5,098,890 is directedto antisense oligonucleotides complementary to the c-myb oncogene andantisense oligonucleotide therapies for certain cancerous conditions.U.S. Pat. No. 5,087,617 provides methods for treating cancer patientswith antisense oligonucleotides. U.S. Pat. No. 5,166,195 providesoligonucleotide inhibitors of Human Immunodeficiency Virus (HIV). U.S.Pat. No. 5,004,810 provides oligomers capable of hybridizing to herpessimplex virus Vmw65 mRNA and inhibiting replication. U.S. Pat. No.5,194,428 provides antisense oligonucleotides having antiviral activityagainst influenzavirus. U.S. Pat. No. 4,806,463 provides antisenseoligonucleotides and methods using them to inhibit HTLV-III replication.U.S. Pat. No. 5,286,717 provides oligonucleotides having a complementarybase sequence to a portion of an oncogene. U.S. Pat. No. 5,276,019 andU.S. Pat. No. 5,264,423 are directed to phosphorothioate oligonucleotideanalogs used to prevent replication of foreign nucleic acids in cells.U.S. Pat. No. 4,689,320 is directed to antisense oligonucleotides asantiviral agents specific to cytomegalovirus (CMV). U.S. Pat. No.5,098,890 provides oligonucleotides complementary to at least a portionof the mRNA transcript of the human c-myb gene. U.S. Pat. No. 5,242,906provides antisense oligonucleotides useful in the treatment of latentEpstein-Barr virus (EBV) infections. Other examples of antisenseoligonucleotides are provided herein.

Further, oligonucleotides used in the compositions of the presentinvention may be directed to modify the effects of mRNAs or DNAsinvolved in the synthesis of proteins that regulate adhesion of whiteblood cells and to other cell types. The adherence of white blood cellsto vascular endothelium appears to be mediated in part if not in toto byfive cell adhesion molecules ICAM-1, ICAM-2, ELAM-1, VCAM-1 and GMP-140.Dustin and Springer, J. Cell. Biol. 1987, 107, 321. Such antisenseoligonucleotides are designed to hybridize either directly to the mRNAor to a selected DNA portion encoding intercellular adhesion molecule-1(ICAM-1), endothelial leukocyte adhesion molecule-1 (ELAM-1, orE-selectin), and vascular cell adhesion molecule-1 (VCAM-1) as disclosedin U.S. Pat. No. 5,514,788 (Bennett et al., May 7, 1996) and U.S. Pat.No. 5,591,623 (Bennett et al., Jan. 7, 1997), and pending U.S. patentapplication Ser. No. 08/440,740 (filed May 12, 1995) and Ser. No.09/062,416 (filed Apr. 17, 1998). These oligonucleotides have been foundto modulate the activity of the targeted mRNA or DNA, leading to themodulation of the synthesis and metabolism of specific cell adhesionmolecules, and thereby result in palliative and therapeutic effects.Inhibition of ICAM-1, VCAM-1 and ELAM-1 expression is expected to beuseful for the treatment of inflammatory diseases, diseases with aninflammatory component, allograft rejection, psoriasis and other skindiseases, inflammatory bowel disease, cancers and their metastases, andviral infection. Methods of modulating cell adhesion comprisingcontacting the animal with an oligonucleotide composition of the presentinvention are provided.

The antisense compounds used as exemplary biologically active nucleicacids in the studies detailed herein are as follows:

ISIS 2302 is a 2′-deoxyoligonucleotide having a phosphorothioatebackbone and the sequence 5′-GCC-CAA-GCT-GGC-ATC-CGT-CA-3′ (SEQ IDNO:1). ISIS 2302 is targeted to the 3′-untranslated region (3′-UTR) ofthe human ICAM-1 gene. ISIS 2302 is described in U.S. Pat. Nos.5,514,788 and 5,591,623, hereby incorporated by reference.

ISIS 15839 is a phosphorothioate isosequence “hemimer” derivative ofISIS 2302 having the structure 5′-GCC-CAA-GCT-GGC-ATC-CGT-CA-3′ (SEQ IDNO:1), wherein emboldened “C” residues have 5-methylcytosine (m5c) basesand wherein the emboldened, double-underlined residues further comprisea 2′-methoxyethoxy modification (other residues are 2′-deoxy). ISIS15839 is described in co-pending U.S. patent application Ser. No.09/062,416, filed Apr. 17, 1998, hereby incorporated by reference.

ISIS 1939 is a 2′-deoxyoligonucleotide having a phosphorothioatebackbone and the sequence 5′-CCC-CCA-CCA-CTT-CCC-CTC-TC-3′ (SEQ IDNO:2). ISIS 1939 is targeted to the 3′-untranslated region (3′-UTR) ofthe human ICAM-1 gene. ISIS 1939 is described in U.S. Pat. Nos.5,514,788 and 5,591,623, hereby incorporated by reference.

Examination of the predicted RNA secondary structure of the human ICAM-1mRNA 3′-untranslated region (M. Zuker, Science 1989, 244, 48)surprisingly suggested that both ISIS 1939 and ISIS 2302 hybridize tosequences predicted to be in a stable stem-loop structure of the mRNA.Current dogma suggests that when designing antisense oligonucleotidesregions of RNA secondary structure should be avoided. Thus, ISIS 1939and ISIS 2302 would not have been predicted to inhibit ICAM-1expression.

ISIS 2302 (SEQ ID NO: 1) has been found to inhibit ICAM-1 expression inhuman umbilical vein cells, human lung carcinoma cells (A549), humanepidermal carcinoma cells (A431), and human keratinocytes. ISIS 2302 hasalso demonstrated specificity for its target ICAM-1 over other potentialnucleic acid targets such as HLA-A and HLA-B. ISIS 1939 (SEQ ID NO:2)and ISIS 2302 markedly reduced ICAM-1 expression, as detected bynorthern blot analysis to determine mRNA levels, in C8161 human melanomacells. In an experimental metastasis assay, ISIS 2302 decreased themetastatic potential of C8161 cells, and eliminated the enhancedmetastatic ability of C8161 cells resulting from TNF-α treatment. ISIS2302 has also shown significant biological activity in animal models ofinflammatory disease. The data from animal testing has revealed stronganti-inflammatory effects of ISIS 2302 in a number of inflammatorydiseases including Crohn's disease, rheumatoid arthritis, psoriasis,ulcerative colitis, and kidney transplant rejection. When tested onhumans, ISIS 2302 has shown good safety and activity against Crohn'sdisease. Further ISIS 2302 has demonstrated a statistically significantsteroid-sparing effect on treated subjects such that even after fivemonths post-treatment subjects have remained weaned from steroids and indisease remission. This is a surprising and significant finding of ISIS2302's effects.

The oligonucleotides used in the compositions of the present inventionpreferably comprise from about 8 to about 30 nucleotides. It is morepreferred that such oligonucleotides comprise from about 10 to about 25nucleotides.

Antisense oligonucleotides employed in the compositions of the presentinvention may also be used to determine the nature, function andpotential relationship of various genetic components of the body tonormal or abnormal body states of animals. Heretofore, the function of agene has been chiefly examined by the construction of loss-of-functionmutations in the gene (i.e., “knock-out” mutations) in an animal (e.g.,a transgenic mouse). Such tasks are difficult, time-consuming and cannotbe accomplished for genes essential to animal development since the“knock-out” mutation would produce a lethal phenotype. Moreover, theloss-of-function phenotype cannot be transiently introduced during aparticular part of the animal's life cycle or disease state; the“knock-out” mutation is always present. “Antisense knockouts,” that is,the selective modulation of expression of a gene by antisenseoligonucleotides, rather than by direct genetic manipulation, overcomesthese limitations (see, for example, Albert et al., Trends inPharmacological Sciences, 1994, 15, 250). In addition, some genesproduce a variety of mRNA transcripts as a result of processes such asalternative splicing; a “knock-out” mutation typically removes all formsof mRNA transcripts produced from such genes and thus cannot be used toexamine the biological role of a particular mRNA transcript. Byproviding compositions and methods for the simple non-parenteraldelivery of oligonucleotides and other nucleic acids, the presentinvention overcomes these and other shortcomings.

Specific examples of some preferred modified oligonucleotides envisionedfor use in the compositions of the present invention includeoligonucleotides containing modified backbones or non-natural intersugarlinkages. As defined in this specification, oligonucleotides havingmodified backbones include those that retain a phosphorus atom in thebackbone and those that have an atom (or group of atoms) other than aphosphorus atom in the backbone. For the purposes of this specification,and as sometimes referenced in the art, modified oligonucleotides thatdo not have a phosphorus atom in their intersugar backbone, includingpeptide nucleic acids (PNAs) are also be considered to beoligonucleotides.

Specific oligonucleotide chemical modifications are described in thefollowing subsections. It is not necessary for all positions in a givencompound to be uniformly modified, and in fact more than one of thefollowing modifications may be incorporated in a single antisensecompound or even in a single residue thereof, for example, at a singlenucleoside within an oligonucleotide.

A. Modified Linkages:

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalklyphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus atom containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,625,050; and 5,697,248, certain of which arecommonly owned with this application, and each of which is hereinincorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein (i.e., oligonucleosides) have backbones that areformed by short chain alkyl or cycloalkyl intersugar linkages, mixedheteroatom and alkyl or cycloalkyl intersugar linkages, or one or moreshort chain heteroatomic or heterocyclic intersugar linkages. Theseinclude those having morpholino linkages (formed in part from the sugarportion of a nucleoside); siloxane backbones; sulfide, sulfoxide andsulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene containing backbones;sulfamate backbones; methyleneimino and methylenehydrazino backbones;sulfonate and sulfonamide backbones; amide backbones; and others havingmixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, certain of which are commonly ownedwith this application, and each of which is herein incorporated byreference.

In other preferred oligonucleotide mimetics, both the sugar and theintersugar linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497.

Some preferred embodiments of the present invention may employoligonucleotides with phosphorothioate backbones and oligonucleotideswith heteroatom backbones, and in particular —CH₂—NH—O—CH₂—,—CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone],—CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂—[wherein the native phosphodiester backbone is represented as—O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and theamide backbones of the above referenced U.S. Pat. No. 5,602,240. Alsopreferred are oligonucleotides having morpholino backbone structures ofthe above-referenced U.S. Pat. No. 5,034,506.

B. Modified Nucleobases:

The oligonucleotides employed in the compositions of the presentinvention may additionally or alternatively comprise nucleobase (oftenreferred to in the art simply as “base”) modifications or substitutions.As used herein, “unmodified” or “natural” nucleobases include the purinebases adenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in the Concise Encyclopedia Of PolymerScience And Engineering, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., AngewandteChemie, International Edition, 1991, 30, 613, and those disclosed bySanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain ofthese nucleobases are particularly useful for increasing the bindingaffinity of the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2EC (Id., pages276-278) and are presently preferred base substitutions, even moreparticularly when combined with 2′-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; and 5,681,941, certain of which are commonlyowned, and each of which is herein incorporated by reference, andcommonly owned U.S. patent application Ser. No. 08/762,488, filed onDec. 10, 1996, also herein incorporated by reference.

C. Sugar Modifications:

The oligonucleotides employed in the compositions of the presentinvention may additionally or alternatively comprise one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O—, S—, or N-alkyl, O—, S—, orN-alkenyl, or O, S- or N-alkynyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. A preferred modificationincludes 2′-methoxyethoxy [2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE] (Martin et al., Helv. Chim. Acta, 1995,78, 486), i.e., an alkoxyalkoxy group. A further preferred modificationincludes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, alsoknown as 2′-DMAOE, as described in co-owned U.S. patent application Ser.No. 09/016,520, filed on Jan. 30, 1998, the contents of which are hereinincorporated by reference.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugars structures include, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,0531 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, certain of which are commonlyowned, and each of which is herein incorporated by reference, andcommonly owned U.S. patent application Ser. No. 08/468,037, filed onJun. 5, 1995, also herein incorporated by reference.

D. Other Modifications:

Additional modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide and the 5′ position of 5′ terminal nucleotide. Forexample, one additional modification of the oligonucleotides employed inthe compositions of the present invention involves chemically linking tothe oligonucleotide one or more moieties or conjugates which enhance theactivity, cellular distribution or cellular uptake of theoligonucleotide. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4, 1053), a thioether, e.g., hexyl-5-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3, 2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327;Svinarchuk et al., Biochimie, 1993, 75, 49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923).

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned, and each of which is herein incorporated byreference.

A preferred conjugate imparting improved absorption of oligonucleotidesin the gut is folic acid. Accordingly, there is provided a compositionfor oral administration comprising an oligonucleotide and a carrierwherein said oligonucleotide is conjugated to folic acid. Folic acid(folate) may be conjugated to the 3′ or 5′ termini of oligonucleotides,to a nucleobase or to a 2′ position of any of the sugar residues in thechain. Conjugation may be via any suitable chemical linker utilizingfunctional groups on the oligonucleotide and folate.Oligonucleotide-folate conjugates and methods in preparing are describedin copending U.S. patent application Ser. No. 09/098,166 (filed Jun. 16,1998) and Ser. No. 09/275,505 (filed Mar. 24, 1999) both incorporatedherein by reference.

E. Chimeric Oligonucleotides:

The present invention also includes compositions employing antisensecompounds which are chimeric compounds. “Chimeric” antisense compoundsor “chimeras,” in the context of this invention, are antisensecompounds, particularly oligonucleotides, which contain two or morechemically distinct regions, each made up of at least one monomer unit,i.e., a nucleotide in the case of an oligonucleotide compound. Theseoligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioateoligodeoxynucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart. RNase H-mediated target cleavage is distinct from the use ofribozymes to cleave nucleic acids.

By way of example, such “chimeras” may be “gapmers,” i.e.,oligonucleotides in which a central portion (the “gap”) of theoligonucleotide serves as a substrate for, e.g., RNase H, and the 5′ and3′ portions (the “wings”) are modified in such a fashion so as to havegreater affinity for, or stability when duplexed with, the target RNAmolecule but are unable to support nuclease activity (e.g., 2′-fluoro-or 2′-methoxyethoxy-substituted). Other chimeras include “hemimers,”that is, oligonucleotides in which the 5′ portion of the oligonucleotideserves as a substrate for, e.g., RNase H, whereas the 3′ portion ismodified in such a fashion so as to have greater affinity for, orstability when duplexed with, the target RNA molecule but is unable tosupport nuclease activity (e.g., 2′-fluoro- or2′-methoxyethoxy-substituted), or vice-versa.

A number of chemical modifications to oligonucleotides that confergreater oligonucleotide:RNA duplex stability have been described byFreier et al. (Nucl. Acids Res., 1997, 25, 4429). Such modifications arepreferred for the RNase H-refractory portions of chimericoligonucleotides and may generally be used to enhance the affinity of anantisense compound for a target RNA.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned, and each of which is hereinincorporated by reference, and commonly owned and allowed U.S. patentapplication Ser. No. 08/465,880, filed on Jun. 6, 1995, also hereinincorporated by reference.

The present invention also includes compositions employingoligonucleotides that are substantially chirally pure with regard toparticular positions within the oligonucleotides. Examples ofsubstantially chirally pure oligonucleotides include, but are notlimited to, those having phosphorothioate linkages that are at least 75%Sp or Rp (Cook et al., U.S. Pat. No. 5,587,361) and those havingsubstantially chirally pure (Sp or Rp) alkylphosphonate, phosphoamidateor phosphotriester linkages (Cook, U.S. Pat. Nos. 5,212,295 and5,521,302).

The present invention further encompasses compositions employingribozymes. Synthetic RNA molecules and derivatives thereof that catalyzehighly specific endoribonuclease activities are known as ribozymes.(See, generally, U.S. Pat. No. 5,543,508 to Haseloff et al., issued Aug.6, 1996, and U.S. Pat. No. 5,545,729 to Goodchild et al., issued Aug.13, 1996.) The cleavage reactions are catalyzed by the RNA moleculesthemselves. In naturally occurring RNA molecules, the sites ofself-catalyzed cleavage are located within highly conserved regions ofRNA secondary structure (Buzayan et al., Proc. Natl. Acad. Sci. U.S.A.,1986, 83, 8859; Forster et al., Cell, 1987, 50, 9). Naturally occurringautocatalytic RNA molecules have been modified to generate ribozymeswhich can be targeted to a particular cellular or pathogenic RNAmolecule with a high degree of specificity. Thus, ribozymes serve thesame general purpose as antisense oligonucleotides (i.e., modulation ofexpression of a specific gene) and, like oligonucleotides, are nucleicacids possessing significant portions of single-strandedness. That is,ribozymes have substantial chemical and functional identity witholigonucleotides and are thus considered to be equivalents for purposesof the present invention.

Other biologically active oligonucleotides may be formulated in thecompositions of the invention and used for therapeutic, palliative orprophylactic purposes according to the methods of the invention. Suchother biologically active oligonucleotides include, but are not limitedto, antisense compounds including, inter alia, antisenseoligonucleotides, antisense PNAs and ribozymes (described supra) andEGSs, as well as aptamers and molecular decoys (described infra).

Sequences that recruit RNase P are known as External Guide Sequences,hence the abbreviation “EGS.” EGSs are antisense compounds that directof an endogenous nuclease (RNase P) to a targeted nucleic acid (Forsteret al., Science, 1990, 249, 783; Guerrier-Takada et al., Proc. Natl.Acad. Sci. USA, 1997, 94, 8468).

Antisense compounds may alternatively or additionally comprise asynthetic moiety having nuclease activity covalently linked to anoligonucleotide having an antisense sequence instead of relying uponrecruitment of an endogenous nuclease. Synthetic moieties havingnuclease activity include, but are not limited to, enzymatic RNAs (as inribozymes), lanthanide ion complexes, and the like (Haseloff et al.,Nature, 1988, 334, 585; Baker et al., J. Am. Chem. Soc., 1997, 119,8749).

Aptamers are single-stranded oligonucleotides that bind specific ligandsvia a mechanism other than Watson-Crick base pairing. Aptamers aretypically targeted to, e.g., a protein and are not designed to bind to anucleic acid (Ellington et al., Nature, 1990, 346, 818).

Molecular decoys are short double-stranded nucleic acids (includingsingle-stranded nucleic acids designed to “fold back” on themselves)that mimic a site on a nucleic acid to which a factor, such as aprotein, binds. Such decoys are expected to competitively inhibit thefactor; that is, because the factor molecules are bound to an excess ofthe decoy, the concentration of factor bound to the cellular sitecorresponding to the decoy decreases, with resulting therapeutic,palliative or prophylactic effects. Methods of identifying andconstructing nucleic acid decoy molecules are described in, e.g., U.S.Pat. No. 5,716,780 to Edwards et al.

Another type of bioactive oligonucleotide is an RNA-DNA hybrid moleculethat can direct gene conversion of an endogenous nucleic acid(Cole-Strauss et al., Science, 1996, 273, 1386).

Examples of specific oligonucleotides and the target genes to which theyinhibit, which may be employed in formulations of the present inventioninclude:

(SEQ ID NO: 1) ISIS-2302 GCCCA AGCTG GCATC CGTCA ICAM-1 (SEQ ID NO: 1)ISIS-15839 GCCCA AGCTG GC AT C   C GT C A ICAM-1 (SEQ ID NO: 2)ISIS-1939 CCCCC ACCAC TTCCC CTCTC ICAM-1 (SEQ ID NO: 48) ISIS-2922GCGTT TGCTC TTCTT CTTGC G HCMV (SEQ ID NO: 48) ISIS-13312 G C GTT TGCTC TTCTT  C TTG C  G HCMV (SEQ ID NO: 49) ISIS-3521GTTCT CGCTG GTGAG TTTCA PKCα (SEQ ID NO: 49) ISIS-9605 GTT C T  CGCTG GTGAG TTT C A PKCα (SEQ ID NO: 49) ISIS-9606 GTT C T  CGCTG GTGAG TTT C A PKCα (SEQ ID NO: 50) ISIS-14859 AACTT GTG C T TG C TC PKCα (SEQ ID NO: 16) ISIS-2503 TCCGT CATCG CTCCT CAGGG Ha-ras(SEQ ID NO: 19) ISIS-5132 TCCCG CCTGT GACAT GCATT c-raf (SEQ ID NO: 51)ISIS-14803 GTGCT CATGG TGCAC GGTCT HCV (SEQ ID NO: 52) ISIS-28089GTGTG CCAGA CACCC TAT C T TNFα (SEQ ID NO: 53) ISIS-104838 G CTGA TTAGA GAGAG GT CCC TNFα (SEQ ID NO: 54) ISIS-2105TTGCT TCCAT CTTCC TCGTC HPVwherein (i) each oligo backbone linkage is a phosphorothioate linkage(except ISIS-9605) and (ii) each sugar is 2′-deoxy unless represented inbold font in which case it incorporates a 2′-O-methoxyethyl group andiii) underlined cytosine nucleosides incorporate a 5-methyl substituenton their nucleobase. ISIS-9605 incorporates natural phosphodiester bondsat the first five and last five linkages with the remainder beingphosphorothioate linkages.

F. Synthesis:

The oligonucleotides used in the compositions of the present inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides such as thephosphorothioates and alkylated derivatives.

1. Teachings

regarding the synthesis of particular modified oligonucleotides may befound in the following U.S. patents or pending patent applications, eachof which is commonly assigned with this application: U.S. Pat. Nos.5,138,045 and 5,218,105, drawn to polyamine conjugated oligonucleotides;U.S. Pat. No. 5,212,295, drawn to monomers for the preparation ofoligonucleotides having chiral phosphorus linkages; U.S. Pat. Nos.5,378,825 and 5,541,307, drawn to oligonucleotides having modifiedbackbones; U.S. Pat. No. 5,386,023, drawn to backbone modifiedoligonucleotides and the preparation thereof through reductive coupling;U.S. Pat. No. 5,457,191, drawn to modified nucleobases based on the3-deazapurine ring system and methods of synthesis thereof; U.S. Pat.No. 5,459,255, drawn to modified nucleobases based on N-2 substitutedpurines; U.S. Pat. No. 5,521,302, drawn to processes for preparingoligonucleotides having chiral phosphorus linkages; U.S. Pat. No.5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746,drawn to oligonucleotides having β-lactam backbones; U.S. Pat. No.5,571,902, drawn to methods and materials for the synthesis ofoligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides havingalkylthio groups, wherein such groups may be used as linkers to othermoieties attached at any of a variety of positions of the nucleoside;U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides havingphosphorothioate linkages of high chiral purity; U.S. Pat. No.5,506,351, drawn to processes for the preparation of 2′-O-alkylguanosine and related compounds, including 2,6-diaminopurine compounds;U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotideshaving 3-deazapurines; U.S. Pat. No. 5,223,168, issued Jun. 29, 1993,and U.S. Pat. No. 5,608,046, both drawn to conjugated 4′-desmethylnucleoside analogs; U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn tobackbone modified oligonucleotide analogs; and U.S. patent applicationSer. No. 08/383,666, filed Feb. 3, 1995, and U.S. Pat. No. 5,459,255,drawn to, inter alia, methods of synthesizing2′-fluoro-oligonucleotides.

2. Bioequivalents:

The compositions of the present invention encompass any pharmaceuticallyacceptable compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to “prodrugs” and “pharmaceutically acceptablesalts” of the antisense compounds of the invention and otherbioequivalents.

A. Oligonucleotide Prodrugs:

The oligonucleotide and nucleic acid compounds employed in thecompositions of the present invention may additionally or alternativelybe prepared to be delivered in a “prodrug” form. The term “prodrug”indicates a therapeutic agent that is prepared in an inactive form thatis converted to an active form (i.e., drug) within the body or cellsthereof by the action of endogenous enzymes or other chemicals and/orconditions. In particular, prodrug versions of the antisense compoundsmay be prepared as SATE [(S-acetyl-2-thioethyl)phosphate] derivativesaccording to the methods disclosed in WO 93/24510 (Gosselin et al.,published Dec. 9, 1993).

B. Pharmaceutically Acceptable Salts:

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the oligonucleotide and nucleicacid compounds employed in the compositions of the present invention(i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto).

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, ammonium, polyamines such as spermine and spermidine, and thelike. Examples of suitable amines are chloroprocaine, choline,N,N′-dibenzylethylenediamine, diethanolamine, dicyclohexylamine,ethylenediamine, N-methylglucamine, and procaine (see, for example,Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci., 1977, 66:1).The base addition salts of said acidic compounds are prepared bycontacting the free acid form with a sufficient amount of the desiredbase to produce the salt in the conventional manner. The free acid formmay be regenerated by contacting the salt form with an acid andisolating the free acid in the conventional manner. The free acid formsdiffer from their respective salt forms somewhat in certain physicalproperties such as solubility in polar solvents, but otherwise the saltsare equivalent to their respective free acid for purposes of the presentinvention.

During the process of oligonucleotide synthesis, nucleoside monomers areattached to the chain one at a time in a repeated series of chemicalreactions such as nucleoside monomer coupling, oxidation, capping anddetritylation. The stepwise yield for each nucleoside addition is above99%. That means that less than 1% of the sequence chain failed to begenerated from the nucleoside monomer addition in each step as the totalresults of the incomplete coupling followed by the incomplete capping,detritylation and oxidation (Smith, Anal. Chem., 1988, 60, 381A). Allthe shorter oligonucleotides, ranging from (n-1), (n-2), etc., to 1-mers(nucleotides), are present as impurities in the n-mer oligonucleotideproduct. Among the impurities, (n-2)-mer and shorter oligonucleotideimpurities are present in very small amounts and can be easily removedby chromatographic purification (Warren et al., Chapter 9 In: Methods inMolecular Biology, Vol. 26: Protocols for Oligonucleotide Conjugates,Agrawal, S., Ed., 1994, Humana Press Inc., Totowa, N.J., pages 233-264).However, due to the lack of chromatographic selectivity and productyield, some (n-1)-mer impurities are still present in the full-length(i.e., n-mer) oligonucleotide product after the purification process.The (n-1) portion consists of the mixture of all possible single basedeletion sequences relative to the n-mer parent oligonucleotide. Such(n-1) impurities can be classified as terminal deletion or internaldeletion sequences, depending upon the position of the missing base(i.e., either at the 5′ or 3′ terminus or internally). When anoligonucleotide containing single base deletion sequence impurities isused as a drug (Crooke, Hematologic Pathology, 1995, 9, 59), theterminal deletion sequence impurities will bind to the same target mRNAas the full length sequence but with a slightly lower affinity. Thus, tosome extent, such impurities can be considered as part of the activedrug component, and are thus considered to be bioequivalents forpurposes of the present invention.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Thecompositions of the present invention may be formulated into any of manypossible dosage forms such as, but not limited to, tablets, capsules,liquid syrups, soft gels, suppositories, and enemas.

Pharmaceutically acceptable organic or inorganic carrier substancessuitable for non-parenteral administration which do not deleteriouslyreact with nucleic acids can also be used to formulate the compositionsof the present invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like. The formulations can be sterilizedand, if desired, mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings flavorings and/oraromatic substances and the like which do not deleteriously interactwith the nucleic acid(s) of the formulation.

The compositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and formulations containing liposomes.While basically similar in nature these formulations vary in thecomponents and the consistency of the final product. The know-how on thepreparation of such compositions and formulations is generally known tothose skilled in the pharmaceutical and formulation arts and may beapplied to the formulation of the compositions of the present invention.

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically

heterogenous systems of one liquid dispersed in another in the form ofdroplets usually exceeding 0.1 um in diameter. (Idson, in PharmaceuticalDosage Forms: Disperse Systems, Vol. 1, Lieberman, Rieger and Banker,Eds., Marcel Dekker, Inc., New York, N.Y., 1988, p. 199; Rosoff, inPharmaceutical Dosage Forms: Disperse Systems, Vol. 1, Lieberman, Riegerand Banker, Eds., Marcel Dekker, Inc., New York, N.Y., 1988, p. 245;Block, in Pharmaceutical Dosage Forms: Disperse Systems, Vol. 2,Lieberman, Rieger and Banker, Eds., Marcel Dekker, Inc., New York, N.Y.,1988, p. 335; Higuchi et al., in “Remington's Pharmaceutical Sciences,”Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are oftenbiphasic systems comprising of two immiscible liquid phases intimatelymixed and dispersed with each other. In general, emulsions may be eitherwater in oil (w/o) or of the oil in water (o/w) variety. When an aqueousphase is finely divided into and dispersed as minute droplets into abulk oily phase the resulting composition is called a water in oil (w/o)emulsion. Alternatively, when an oily phase is finely divided into anddispersed as minute droplets into a bulk aqueous phase the resultingcomposition is called an oil in water (o/w) emulsion.

Emulsions may contain additional components in addition to the dispersedphases and the active drug which may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, andanti-oxidants may also be present in emulsions as needed. Pharmaceuticalemulsions may also be multiple emulsions that are comprised of more thantwo phases such as, for example, in the case of oil in water in oil(o/w/o) and water in oil in water (w/o/w) emulsions. Such complexformulations often provide certain advantages that simple binaryemulsions do not. Multiple emulsions in which individual oil droplets ofan o/w emulsion enclose small water droplets constitute a w/o/wemulsion. Likewise a system of oil droplets enclosed in globules ofwater stabilized in an oily continuous provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms: Disperse Systems, Vol. 1,Lieberman, Rieger and Banker, Eds., Marcel Dekker, Inc., New York, N.Y.,1988, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage FormsDisperse Systems, Vol. 1, Lieberman, Rieger and Banker, Eds., MarcelDekker, Inc., New York, N.Y., 1988, p. 285; Idson, in PharmaceuticalDosage Forms: Disperse Systems, Vol. 1, Lieberman, Rieger and Banker,Eds., Marcel Dekker, Inc., New York, N.Y., 1988, p. 199). Surfactantsare typically amphiphilic and comprise a hydrophilic and a hydrophobicportion. The ratio of the hydrophilic to the hydrophobic nature of thesurfactant has been termed the hydrophile/lipophile balance (HLB) and isa valuable tool in categorizing and selecting surfactants in thepreparation of formulations. Surfactants may be classified intodifferent classes based on the nature of the hydrophilic group into:nonionic, anionic, cationic and amphoteric (Rieger, in PharmaceuticalDosage Forms: Disperse Systems, Vol. 1, Lieberman, Rieger and Banker,Eds., Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms: Disperse Systems, Vol. 1,Lieberman, Rieger and Banker, Eds., Marcel Dekker, Inc., New York, N.Y.,1988, p. 335; Idson, Id., p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylc cellulose andcarboxypropyl cellulose), and synthetic polymers (for example,carbomers, cellulose ethers, and carboxyvinyl polymers). These disperseor swell in water to form colloidal solutions that stabilize emulsionsby forming strong interfacial films around the dispersed-phase dropletsand by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms DisperseSystems, Vol. 1, Lieberman, Rieger and Banker, Eds., Marcel Dekker,Inc., New York, N.Y., 1988, p. 199). Emulsion formulations for oraldelivery have been very widely used because of reasons of ease offormulation, efficacy from an absorption and bioavailability standpoint.(Rosoff, in Pharmaceutical Dosage Forms: Disperse Systems, Vol. 1,Lieberman, Rieger and Banker, Eds., Marcel Dekker, Inc., New York, N.Y.,1988, p. 245; Idson, Id., p. 199). Mineral-oil base laxatives,oil-soluble vitamins and high fat nutritive preparations are among thematerials that have commonly been administered orally as o/w emulsions.

In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms: DisperseSystems, Vol. 1, Lieberman, Rieger and Banker, Eds., Marcel Dekker,Inc., New York, N.Y., 1988, p. 245). Typically microemulsions aresystems that are prepared by first dispersing an oil in an aqueoussurfactant solution and then adding a sufficient amount of a fourthcomponent, generally an intermediate chain-length alcohol to form atransparent system. Therefore, microemulsions have also been describedas thermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in “Remington's PharmaceuticalSciences,” Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms: Disperse Systems, Vol. 1, Lieberman, Riegerand Banker, Eds., Marcel Dekker, Inc., New York, N.Y., 1988, p. 245;Block, Id., p. 335). Compared to conventional emulsions, microemulsionsoffer the advantage of solubilizing water-insoluble drugs in aformulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138). Often microemulsions may form spontaneously whentheir components are brought together at ambient temperature. This maybe particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. Further advantages are that liposomes obtained from naturalphospholipids are biocompatible and biodegradable, liposomes canincorporate a wide range of water and lipid soluble drugs, liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage FormsDisperse Systems, Vol. 1, Lieberman, Rieger and Banker, Eds., MarcelDekker, Inc., New York, N.Y., 1988, p. 245). Important considerations inthe preparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes. Liposomes can beadministered orally and in aerosols and topical applications.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms: Disperse Systems, Vol. 1, Lieberman, Riegerand Banker, Eds., Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms: DisperseSystems, Vol. 1, Lieberman, Rieger and Banker, Eds., Marcel Dekker,Inc., New York, N.Y., 1988, p. 285).

In one embodiment of the invention, a nucleic acid is administered viathe rectal mode. In particular, compositions for rectal administrationinclude solutions (enemas and suppositories) and emulsions. Rectalsuppositories for adults are usually tapered at one or both ends andtypically weigh about 2 g each, with infant rectal suppositoriestypically weighing about one-half as much, when the usual base, cocoabutter, is used (Block, Chapter 87 In: Remington's PharmaceuticalSciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,1990).

The use of absorption-promoting adjuvants is known in the art for themodification of the barrier function of the rectal membrane and has beenreviewed (Nishihata and Rytting, Advanced Drug Delivery Reviews, 1997,28, 205). Absorption-promoting adjuvants have shown promising effects onthe performance of formulations of poorly absorbed drugs such asmoderately large water-soluble drugs and peptides. Enamine derivativesof amino acids have exhibited absorption promoting properties but themechanism by which they increase rectal absorption is unclear. Compoundssuch as chelating agents, and sulfhydryl depleters have been shown toincrease the rectal absorption of drugs through the paracellular routeas well as the transcellular route. Salicylate and its derivatives alsoincrease absorption of drugs administered via the rectal route via bothparacellular and transcellular paths. Fatty acids show propertiessimilar to salicylates when enhancing rectal absorption of drugs. Lectinis also known to increase rectal absorption of drugs via induction ofmicrovillus infusion.

In a preferred embodiment of the invention, one or more nucleic acidsare administered via oral delivery.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, troches, tablets or SECs (soft elastic capsules or “caplets”).Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids,carrier substances or binders may be desirably added to suchformulations. A tablet may be made by compression or molding, optionallywith one or more accessory ingredients. Compressed tablets may beprepared by compressing in a suitable machine, the active ingredients ina free-flowing form such as a powder or granules, optionally mixed witha binder (PVP or gums such as tragecanth, acacia, carrageenan),lubricant (e.g. stearates such as magnesium stearate), glidant (talc,colloidal silica dioxide), inert diluent, preservative, surface activeor dispersing agent. Preferred binders/disintegrants include EMDEX(dextrate), PRECIROL (triglyceride), PEG, and AVICEL (cellulose). Moldedtablets may be made by molding in a suitable machine a mixture of thepowdered compound moistened with an inert liquid diluent. The tabletsmay optionally be coated or scored and may be formulated so as toprovide slow or controlled release of the active ingredients therein.

The use of such formulations has the effect of delivering the nucleicacid to the alimentary canal for exposure to the mucosa thereof.Accordingly, the formulation can contain an enteric material effectivein protecting the nucleic acid from pH extremes of the stomach, or inreleasing the nucleic acid over time to optimize the delivery thereof toa particular mucosal site. Enteric materials for acid-resistant tablets,capsules and caplets are known in the art and typically include acetatephthalate, propylene glycol, sorbitan monoleate, cellulose acetatephthalate (CAP), cellulose acetate trimellitate and hydroxy propylmethyl cellulose phthalate (HPMCP). Enteric materials may beincorporated within the dosage form or may be a coating substantiallycovering the entire surface of tablets, capsules or caplets. Entericmaterials may also be accompanied by plasticizers which impart flexibleresiliency to the material for resisting fracturing, for example duringtablet curing or aging. Plasticizers are known in the art and typicallyinclude diethyl phthalate (DEP), triacetin, dibutyl sebacate (DBS),dibutyl phthalate (DBP) and triethyl citrate (TEC).

Various methods for producing formulations for alimentary delivery arewell known in the art. See, generally, Nairn, Chapter 83; Block, Chapter87; Rudnic et al., Chapter 89; Porter, Chapter 90; and Longer et al.,Chapter 91 In: Remington's Pharmaceutical Sciences, 18th Ed., German,ed., Mack Publishing Co., Easton, Pa., 1990. The compositions of thisinvention can be converted in a known manner into the customaryformulations, such as tablets, coated tablets, pills, granules,capsules, aerosols, syrups, emulsions, suspensions and solutions, usinginert, non-toxic, pharmaceutically suitable excipients or solvents. Thetherapeutically active compound should in each case be present here in aconcentration of about 0.5% to about 95% by weight of the total mixture,that is to say in amounts which are sufficient to achieve the stateddosage range. Compositions may be formulated in a conventional mannerusing additional pharmaceutically acceptable carriers or excipients asappropriate. Thus, the composition may be prepared by conventional meanswith carriers or excipients such as binding agents (e.g., pregelatinisedmaize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose);fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc or silica);disintegrants (e.g., starch or sodium starch glycolate); or wettingagents (e.g., sodium lauryl sulfate). Tablets may be coated by methodswell known in the art. The preparations may also contain flavoring,coloring and/or sweetening agents as appropriate.

Capsules used for oral delivery may include formulations that are wellknown in the art. Further, multicompartment hard capsules with controlrelease properties as described by Digenis et al., U.S. Pat. No.5,672,359, and water permeable capsules with a multi-stage drug deliverysystem as described by Amidon et al., U.S. Pat. No. 5,674,530 may alsobe used to formulate the compositions of the present invention.

The formulation of pharmaceutical compositions and their subsequentadministration is believed to be within the skill of those in the art.Specific comments regarding the present invention are presented below.

In general, for therapeutic applications, a patient (i.e., an animal,including a human) having or predisposed to a disease or disorder isadministered one or more nucleic acids, including oligonucleotides, inaccordance with the invention in a pharmaceutically acceptable carrierin doses ranging from 0.01 ug to 100 g per kg of body weight dependingon the age of the patient and the severity of the disorder or diseasestate being treated. Further, the treatment regimen may last for aperiod of time which will vary depending upon the nature of theparticular disease or disorder, its severity and the overall conditionof the patient, and may extend from once daily to once every 20 years.In the context of the invention, the term “treatment regimen” is meantto encompass therapeutic, palliative and prophylactic modalities.Following treatment, the patient is monitored for changes in his/hercondition and for alleviation of the symptoms of the disorder or diseasestate. The dosage of the nucleic acid may either be increased if thepatient does not respond significantly to current dosage levels, or thedose may be decreased if an alleviation of the symptoms of the disorderor disease state is observed, or if the disorder or disease state hasbeen abated.

Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀ values found to be effective in invitro and in vivo animal models. In general, dosage is from 0.01 μg to100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. An optimaldosing schedule is used to deliver a therapeutically effective amount ofthe nucleic acid being administered via a particular mode ofadministration.

The term “therapeutically effective amount,” for the purposes of theinvention, refers to the amount of nucleic acid-containing formulationwhich is effective to achieve an intended purpose without undesirableside effects (such as toxicity, irritation or allergic response).Although individual needs may vary, determination of optimal ranges foreffective amounts of formulations is within the skill of the art. Humandoses can be extrapolated from animal studies (Katocs et al., Chapter 27In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990). Generally, the dosage required toprovide an effective amount of a formulation, which can be adjusted byone skilled in the art, will vary depending on the age, health, physicalcondition, weight, type and extent of the disease or disorder of therecipient, frequency of treatment, the nature of concurrent therapy (ifany) and the nature and scope of the desired effect(s) (Nies et al.,Chapter 3 In: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York,N.Y., 1996).

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the nucleic acid is administered in maintenance doses,ranging from 0.01 ug to 100 g per kg of body weight, once or more daily,to once every 20 years. For example, in the case of in individual knownor suspected of being prone to an autoimmune or inflammatory condition,prophylactic effects may be achieved by administration of preventativedoses, ranging from 0.01 ug to 100 g per kg of body weight, once or moredaily, to once every 20 years. In like fashion, an individual may bemade less susceptible to an inflammatory condition that is expected tooccur as a result of some medical treatment, e.g., graft versus hostdisease resulting from the transplantation of cells, tissue or an organinto the individual.

Formulations for non-parenteral administration of nucleic acids mayinclude sterile and non-sterile aqueous solutions, non-aqueous solutionsin common solvents such as alcohols, or solutions of the nucleic acidsin liquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic carrier substances suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used. Suitable pharmaceutically acceptable carriers include, but arenot limited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike. The formulations can be sterilized and, if desired, mixed withauxiliary agents, e.g., lubricants, preservatives, stabilizers, wettingagents, emulsifiers, salts for influencing osmotic pressure, buffers,colorings flavorings and/or aromatic substances and the like which donot deleteriously interact with the nucleic acid(s) of the formulation.Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

The pharmaceutical formulations, which may conveniently be presented inunit dosage form, may be prepared according to conventional techniqueswell known in the pharmaceutical industry. Such techniques include thestep of bringing into association the active ingredients with thepharmaceutical carrier(s) or excipient(s). In general the formulationsare prepared by uniformly and intimately bringing into association theactive ingredients with liquid carriers or finely divided solid carriersor both, and then, if necessary, shaping the product.

A number of bioequivalents of oligonucleotides and other nucleic acidsmay also be employed in accordance with the present invention. Theinvention therefore, also encompasses oligonucleotide and nucleic acidequivalents such as, but not limited to, prodrugs of oligonucleotidesand nucleic acids, deletion derivatives, conjugates of oligonucleotides,aptamers, and ribozymes.

The methods and compositions of the present invention also encompass themyriad deletion oligonucleotides, both internal and terminal deletionoligonucleotides, that are synthesized during the process of solid-phasemanufacture of oligonucleotides for such deletion sequences are for allpractical purposes bioequivalents. Synthetic RNA molecules and theirderivatives that possess specific catalytic activities are known asribozymes and are also considered bioequivalents of oligonucleotides forthe purposes of the methods and compositions of the present invention.Also considered bioequivalents of oligonucleotides, for the purposes ofthe methods and compositions of the present invention, are peptidenucleic acids (PNAs) and aptamers (see, generally, Ellington et al.,Nature, 1990, 346, 818; U.S. Pat. No. 5,523,389 (Ecker et al., Jun. 4,1996)).

The name aptamer has been coined by Ellington and Szostak (Nature, 1990,346, 818) for nucleic acid molecules that fit and therefore bind withsignificant specificity to non-nucleic acid ligands such as peptides,proteins and small molecules such as drugs and dyes. Because of thesespecific ligand binding properties, nucleic acids and oligonucleotidesthat may be classified as aptamers may be readily purified or isolatedvia affinity chromatography using columns that bear immobilized ligand.Aptamers may be nucleic acids that are relatively short to those thatare as large as a few hundred nucleotides. For example, Ellington andSzostak have reported the discovery of RNA aptamers that are 155nucleotides long and that bind dyes such as Cibacron Blue and ReactiveBlue 4 (Ellington and Szostak, Nature, 1990, 346, 818) with very goodselectivity. While RNA molecules were first referred to as aptamers, theterm as used in the present invention refers to any nucleic acid oroligonucleotide that exhibits specific binding to small molecule ligandsincluding, but not limited to, DNA, RNA, DNA derivatives and conjugates,RNA derivatives and conjugates, modified oligonucleotides, chimericoligonucleotides, and gapmers.

The invention is drawn to the non-parenteral administration of a nucleicacid, such as an oligonucleotide, having biological activity, to ananimal. By “having biological activity,” it is meant that the nucleicacid functions to modulate the expression of one or more genes in ananimal as reflected in either absolute function of the gene (such asribozyme activity) or by production of proteins coded by such genes. Inthe context of this invention, “to modulate” means to either effect anincrease (stimulate) or a decrease (inhibit) in the expression of agene. Such modulation can be achieved by, for example, an antisenseoligonucleotide by a variety of mechanisms known in the art, includingbut not limited to transcriptional arrest; effects on RNA processing(capping, polyadenylation and splicing) and transportation; enhancementor reduction of cellular degradation of the target nucleic acid; andtranslational arrest (Crooke et al., Exp. Opin. Ther. Patents, 1996, 6,1).

In an animal other than a human, the compositions and methods of theinvention can be used to study the function of one or more genes in theanimal. For example, antisense oligonucleotides have been systemicallyadministered to rats in order to study the role of theN-methyl-D-aspartate receptor in neuronal death, to mice in order toinvestigate the biological role of protein kinase C-a, and to rats inorder to examine the role of the neuropeptide Y1 receptor in anxiety(Wahlestedt et al., Nature, 1993, 363, 260; Dean et al., Proc. Natl.Acad. Sci. U.S.A., 1994, 91, 11762; and Wahlestedt et al., Science,1993, 259, 528, respectively). In instances where complex families ofrelated proteins are being investigated, “antisense knockouts” (i.e.,inhibition of a gene by systemic administration of antisenseoligonucleotides) may represent the most accurate means for examining aspecific member of the family (see, generally, Albert et al., TrendsPharmacol. Sci., 1994, 15, 250).

As stated, the compositions and methods of the invention are usefultherapeutically, i.e., to provide therapeutic, palliative orprophylactic relief to an animal, including a human, having or suspectedof having or of being susceptible to, a disease or disorder that istreatable in whole or in part with one or more nucleic acids. The term“disease or disorder” (1) includes any abnormal condition of an organismor part, especially as a consequence of infection, inherent weakness,environmental stress, that impairs normal physiological functioning; (2)excludes pregnancy per se but not autoimmune and other diseasesassociated with pregnancy; and (3) includes cancers and tumors. The term“having or suspected of having or of being susceptible to” indicatesthat the subject animal has been determined to be, or is suspected ofbeing, at increased risk, relative to the general population of suchanimals, of developing a particular disease or disorder as hereindefined. For example, a subject animal could have a personal and/orfamily medical history that includes frequent occurrences of aparticular disease or disorder. As another example, a subject animalcould have had such a susceptibility determined by genetic screeningaccording to techniques known in the art (see, e.g., U.S. Congress,Office of Technology Assessment, Chapter 5 In: Genetic Monitoring andScreening in the Workplace, OTA-BA-455, U.S. Government Printing Office,Washington, D.C., 1990, pages 75-99). The term “a disease or disorderthat is treatable in whole or in part with one or more nucleic acids”refers to a disease or disorder, as herein defined, (1) the management,modulation or treatment thereof, and/or (2) therapeutic, palliativeand/or prophylactic relief therefrom, can be provided via theadministration of more nucleic acids. In a preferred embodiment, such adisease or disorder is treatable in whole or in part with an antisenseoligonucleotide.

EXAMPLES

The following examples illustrate the invention and are not intended tolimit the same. Those skilled in the art will recognize, or be able toascertain through routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of the present invention.

Example 1 Preparation of Oligonucleotides

A. General Synthetic Techniques:

Oligonucleotides were synthesized on an automated DNA synthesizer usingstandard phosphoramidite chemistry with oxidation using iodine.Beta-cyanoethyldiisopropyl phosphoramidites were purchased from AppliedBiosystems (Foster City, Calif.). For phosphorothioate oligonucleotides,the standard oxidation bottle was replaced by a 0.2 M solution of3H-1,2-benzodithiole-3-one-1,1-dioxide in acetonitrile for the stepwisethiation of the phosphite linkages.

The synthesis of 2′-O-methyl-(T-methoxy-) phosphorothioateoligonucleotides is according to the procedures set forth abovesubstituting 2′-O-methyl b-cyanoethyldiisopropyl phosphoramidites(Chemgenes, Needham, Mass.) for standard phosphoramidites and increasingthe wait cycle after the pulse delivery of tetrazole and base to 360seconds.

Similarly, 2′-O-propyl-(a.k.a 2′-propoxy-) phosphorothioateoligonucleotides are prepared by slight modifications of this procedureand essentially according to procedures disclosed in U.S. patentapplication Ser. No. 08/383,666, filed Feb. 3, 1995, which is assignedto the same assignee as the instant application and which isincorporated by reference herein.

The 2′-fluoro-phosphorothioate oligonucleotides of the invention aresynthesized using 5′-dimethoxytrityl-3′-phosphoramidites and prepared asdisclosed in U.S. patent application Ser. No. 08/383,666, filed Feb. 3,1995, and U.S. Pat. No. 5,459,255, which issued Oct. 8, 1996, both ofwhich are assigned to the same assignee as the instant application andwhich are incorporated by reference herein. The2′-fluoro-oligonucleotides are prepared using phosphoramidite chemistryand a slight modification of the standard DNA synthesis protocol (i.e.,deprotection was effected using methanolic ammonia at room temperature).

PNA antisense analogs are prepared essentially as described in U.S. Pat.Nos. 5,539,082 and 5,539,083, both of which (1) issued Jul. 23, 1996,(2) are assigned to the same assignee as the instant application and (3)are incorporated by reference herein.

Oligonucleotides comprising 2,6-diaminopurine are prepared usingcompounds described in U.S. Pat. No. 5,506,351 which issued Apr. 9,1996, and which is assigned to the same assignee as the instantapplication and incorporated by reference herein, and materials andmethods described by Gaffney et al. (Tetrahedron, 1984, 40, 3), Cholletet al., (Nucl. Acids Res., 1988, 16, 305) and Prosnyak et al. (Genomics,1994, 21, 490). Oligonucleotides comprising 2,6-diaminopurine can alsobe prepared by enzymatic means (Bailly et al., Proc. Natl. Acad. Sci.U.S.A., 1996, 93, 13623).

2′-Methoxyethoxy oligonucleotides of the invention are synthesizedessentially according to the methods of Martin et al. (Helv. Chim. Acta,1995, 78, 486).

B. Oligonucleotide Purification:

After cleavage from the controlled pore glass (CPG) column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide, at 55° C.for 18 hours, the oligonucleotides were purified by precipitation 2×from 0.5 M NaCl with 2.5 volumes of ethanol followed by furtherpurification by reverse phase high liquid pressure chromatography(HPLC). Analytical gel electrophoresis was accomplished in 20%acrylamide, 8 M urea and 45 mM Tris-borate buffer (pH 7).

C. Oligonucleotide Labeling:

Antisense oligonucleotides were labeled in order to detect the presenceof and/or measure the quantity thereof in samples taken during thecourse of the in vivo pharmacokinetic studies described herein. Althoughradiolabeling by tritium exchange is one preferred means of labelingantisense oligonucleotides for such in vivo studies, a variety of othermeans are available for incorporating a variety of radiological,chemical or enzymatic labels into oligonucleotides and other nucleicacids.

1. Tritium Exchange:

Essentially, the procedure of Graham et al. (Nucleic Acids Research,1993, 21, 3737) was used to label oligonucleotides by tritium exchange.Specifically, about 24 mg of oligonucleotide was dissolved in a mixtureof 200 μL of sodium phosphate buffer (pH 7.8), 400 μL of 0.1 mM EDTA (pH8.3) and 200 μL of deionized water. The pH of the resulting mixture wasmeasured and adjusted to pH 7.8 using 0.095 N NaOH. The mixture waslyophilized overnight in a 1.25 mL gasketed polypropylene vial. Theoligonucleotide was dissolved in 8.25 μL of b-mercaptoethanol, whichacts as a free radical scavenger (Graham et al., Nucleic Acids Research,1993, 21, 3737), and 400 μL of tritiated H₂O (5 Ci/gram). The tube wascapped, placed in a 90EC oil bath for 9 hours without stirring, and thenbriefly centrifuged to remove any condensate from the inside lid of thetube. (As an optional analytical step, two 10 μL aliquots (one for HPLCanalysis, one for PAGE analysis) were removed from the reaction tube;each aliquot was added to a separate 1.5 mL standard microfuge tubecontaining 490 μL of 50 uM sodium phosphate buffer (pH 7.8).) Theoligonucleotide mixture is then frozen in liquid nitrogen andtransferred to a lyophilization apparatus wherein lyophilization wascarried out under high vacuum, typically for 3 hours. The material wasthen resuspended in mL of double-distilled H₂O and allowed to exchangefor 1 hour at room temperature. After incubation, the mixture was againquick frozen and lyophilized overnight. (As an optional analytical step,about 1 mg of the oligonucleotide material is removed for HPLCanalysis.) Three further lyophilizations were carried out, each withapproximately 1 mL of double-distilled H₂O, to ensure the removal of anyresidual, unincorporated tritium. The final resuspended oligonucleotidesolution is transferred to a clean polypropylene vial and assayed. Thetritium labeled oligonucleotide is stored at about −70EC.

2. Other Means of Labeling Nucleic Acids:

As is well known in the art, a variety of means are available to labeloligonucleotides and other nucleic acids and to separate unincorporatedlabel from the labeled nucleic acid. For example, double-strandednucleic acids can be radiolabeled by nick translation and primerextension, and a variety of nucleic acids, including oligonucleotides,can be terminally radiolabeled by the use of enzymes such as T4polynucleotide kinase or terminal deoxynucleotidyl transferase (see,generally, Chapter 3 In: Short Protocols in Molecular Biology, 2d Ed.,Ausubel et al., eds., John Wiley & Sons, New York, N.Y., pages 3-11 to3-38; and Chapter 10 In: Molecular Cloning: A Laboratory Manual, 2d Ed.,Sambrook et al., eds., pages 10.1 to 10.70). It is also well known inthe art to label oligonucleotides and other nucleic acids withnonradioactive labels such as, for example, enzymes, fluorescentmoieties and the like (see, for example, Beck, Methods in Enzymology,1992, 216, 143; and Ruth, Chapter 6 In: Protocols for OligonucleotideConjugates (Methods in Molecular Biology, Volume 26) Agrawal, S., ed.,Humana Press, Totowa, N.J., 1994, pages 167-185).

Example 2 Oligonucleotide Targets and Sequences

The present invention is drawn to compositions and formulationscomprising oligonucleotides or nucleic acids and one or more mucosalpenetration enhancers, and methods of using such formulations. In oneembodiment, such formulations are used to study the function of one ormore genes in an animal other than a human. In a preferred embodiment,oligonucleotides are formulated into a pharmaceutical compositionintended for therapeutic delivery to an animal, including a human. Thefollowing tables list, as exemplars, some preferred oligonucleotidesintended for local or systemic therapeutic delivery, as desired, thatmay be administered via non-parenteral means according to thecompositions and methods of the invention. Such desired oligonucleotidesinclude, but are not limited to, those which modulate the expression ofcellular adhesion proteins (Table 1). Other oligonucleotides aredesigned to modulate the rate of cellular proliferation (Table 2), or tohave biological or therapeutic activity against miscellaneous disorders(Table 3) and diseases resulting from eukaryotic pathogens (Table 4),retroviruses including HIV (human immunodeficiency virus; Table 5) andnon-retroviral viruses (Table 6). Further details regarding theseoligonucleotides are provided in the Sequence Listing.

TABLE 1 TARGET OLIGONUCLEOTIDES DESIGNED TO MODULATE CELLULAR ADHESIONCell Surface Commercial or Common Target Name (if any) ofOligonucleotide Sequence Protein Oligonucleotide SEQ ID NO(S): ICAM-1ISIS 2302 1 ICAM-1 ISIS 1939 2 ICAM-1 GM1595 3 VCAM-1 ISIS 5847 4 VCAM-1GM1535 5 ELAM-1 GM1515, GM1516, GM1517 6, 7, 8

TABLE 2 OLIGONUCLEOTIDES DESIGNED TO MODULATE THE RATE OF CELLULARPROLIFERATION Commercial or Common Name (if any) of OligonucleotideSequence Molecular Target Oligonucleotide SEQ ID NO(S): c-myb MYB-AS  9vascular 10, 11, 12 endothelial growth factor (VEGF) bcl-2 13, 14, 15Ha-ras ISIS 2503 16 MRP ISIS 7597 17 A-raf kinase ISIS 9069 18 c-rafkinase ISIS 5132 19

TABLE 3 OLIGONUCLEOTIDES DESIGNED TO HAVE THERAPEUTIC ACTIVITY AGAINSTMISCELLANEOUS DISORDERS Commercial or Common Name (if any) ofOligonucleotide Sequence Disorder Oligonucleotide SEQ ID NO(S):Alzheimer's 20, 21 disease Beta-thalassemia 5'ss & 3'ss 22, 23

TABLE 4 OLIGONUCLEOTIDES DESIGNED TO HAVE THERAPEUTIC ACTIVITY AGAINSTEUKARYOTIC PATHOGENS Commercial or Common Oligonucleotide Name (if any)of Sequence Pathogen/Disease Oligonucleotide SEQ ID NO(S):Plasmodium/malaria PSI, PSII 24, 25 Schistosoma/bloodfluke 26 infections

TABLE 5 OLIGONUCLEOTIDES DESIGNED TO HAVE THERAPEUTIC ACTIVITY AGAINSTRETROVIRUSES, INCLUDING HIV Commercial or Common Oligonucleotide Name(if any) of Sequence Virus/Molecular Target Oligonucleotide SEQ IDNO(S): HIV-1/gag GEM 91 27 HIV-1/gag 28, 29 HIV AR 177 30 HIV/tat, vpr,rev, env, nef 31, 32 HIV/pol, env, vir 33, 34 HIV-1/tat, rev, env, nef35, 36 HIV/gp120 ISIS 5320 37 Hepatitis C virus ISIS 6547 38

TABLE 6 OLIGONUCLEOTIDES DESIGNED TO HAVE THERAPEUTIC ACTIVITY AGAINSTNON-RETROVIRAL VIRUSES Commercial or Common Oligonucleotide Name (ifany) of Sequence Virus/Molecular Target Oligonucleotide SEQ ID NO(S):influenza virus 39, 40 Epstein-Barr Virus 41, 42 Respiratory SyncytialVirus 43, 44 cytomegalovirus (CMV) GEM 132 45 CMV 46, 47 CMV ISIS 292248

Additional oligonucleotides that may be formulated in the compositionsof the invention include, for example, ribozymes, aptamers, moleculardecoys, External Guide Sequences (EGSs) and peptide nucleic acids(PNAs).

Example 3 Evaluation of Formulations by In Situ Perfusion of Rat Ileum

The formulations of the invention may be evaluated as follows.

Methods:

In order to evaluate the formulations, in situ perfusion of rat ileum isperformed essentially according to the procedure of Komiya et al. (Int.J. Pharmaceut., 1980, 4:249). Specifically, male Sprague Dawley ratsweighing 250-300 g are used. After overnight fasting, the rats areanesthetized with 5% pentobarbital (50 mg/kg) by intraperitonealinjection. After a midline abdominal incision is made, the smallintestine is taken out and ileum section located. An incision is made ateach end of a 20 cm ileum segment. The ileum segment is laid out in auniform multiple-S arrangement with 3 bends. The luminal contents of thesection are flushed with buffer. A 10 cm piece of silicon rubber tubingis inserted into the intestinal lumen at each incision and ligated with3-0 silk suture. The proximal end tubing is connected to a 30 mL syringecontaining oligonucleotide solution. The formulation is perfused throughthe intestinal segment by using Sage model 365 syringe pump at 0.125mL/min. The outflow solution is collected in a 2 mL centrifuge tube over5 min intervals for 80 mins. At the end of perfusion study, an aliquotof 0.3 mL blood sample is collected from the portal vein.

The oligonucleotide concentration in the solution before and afterpassing through a 20 cm ileum segment is analyzed by high pressureliquid chromatography (HPLC) while the plasma samples are analyzed bycapillary electrophoresis (CE). In most cases, tritium labeled ISIS 2302is used as a tracer and the radioactivity of solution measured by aliquid scintillation counter. The amount of the drug absorbed from theileum is calculated by dividing the concentration from the average oflast six outflow samples (steady state) to that of the inflow sample.

Example 4 Evaluation of the Bioavailability of Oligonucleotide fromFormulations Following In Vivo (Intrajejunum) Instillation

In order to evaluate the absolute oral bioavailability of ISIS 2302formulations containing various penetration enhancers, in vivointrajejunum instillation was performed with the following formulations(Table 7).

TABLE 7 Intrajejunum Formulations 4a-4c Formulation ISIS 2302 No.Concentration Penetration Enhancer(s) 4a 20 mg/ml DCA 20 mg/ml SolutionCaprate 40 mg/ml Laurate 40 mg/ml 4b 20 mg/ml UDCA 20 mg/ml SolutionCaprate 40 mg/ml Laurate 40 mg/ml 4c 12 mg/ml Microemulsion

Formulation 4a:

First, 100 mg CDCA was transferred to a 5 ml volumetric flask containingabout 3 ml of buffer. The flask was shaken until the CDCA was completelydissolved. Next, 200 mg sodium caprate and 200 mg sodium laurate wereadded to the solution, and the flask was shaken until all of the solidmaterial was completely dissolved. Then, 0.5 ml of ISIS 2302 stocksolution (200 mg/ml) was added to the solution. Finally, the volume ofthe solution was adjusted to 5 ml with buffer.

Formulation 4b:

First, 200 mg sodium caprate and 200 mg sodium laurate were transferredto a 5 ml volumetric flask containing about 3 ml of buffer. Then, 100 mgof UDCA was added and the flask was shaken until the UDCA was completelydissolved. Then, 0.5 ml of ISIS 2302 stock solution (200 mg/ml) wasadded to the solution. Finally, the volume of the solution was adjustedto 5 ml with buffer.

Formulation 4c:

A microemulsion of ISIS 2302 was prepared essentially according to theprocedures of Panayiotis (Pharm. Res., 1984, 11:1385). An aliquot of 0.6ml of ISIS 2302 stock solution (200 mg/ml) was transferred to a 30 mlbeaker containing 1.0 ml of Tween 80 (Sigma Chemical Company St. Louis,Mo.). Next, a mixture of 6.3 ml of Captex 355 (Abitec Corp., Janesville,Wis.) and 2.1 ml of Capmul MCM (Abitec Corp.) was added to the beaker.The resultant solution was stirred until a clear solution was formed.

Methods:

Precannulated Sprague-Dawley rats weighing 250-300 g were used. Afterovernight fasting, the rats were anesthetized with 5% pentobarbital (50mg/kg) by intraperitoneal injection. After a midline abdominal incisionwas made, the small intestine was pulled out and injection site waslocated (2 cm after the ligament of Treitz). An aliquot of 1.0 mL drugsolution was then injected via a 27 gauge needle. The intestine was putback to the body carefully. Muscle was then surgically closed and skinwas clipped after injection. Three hundred μL of blood was collectedfrom a cannula at each sampling time point. The rats were sacrificed inthe CO₂ chamber 24 hours after dosing. Livers and kidneys were excisedand stored at −80EC until analysis. Radioactivity of plasma and tissuesamples were measured. Liver and kidney were also analyzed foroligonucleotide content by capillary gel electrophoresis (CGE).

Results:

The results of study are summarized in Table 8. No significant amount(i.e., ˜0%) of ISIS 2302 (SEQ ID NO:1) was absorbed when a controlsolution (i.e., one lacking any penetration enhancers) was used. Incontrast, when formulated as a solution containing a mixture ofpenetration enhancers (Formulations 4a and 4b) the absolutebioavailability of ISIS 2302 was in the range of 8 to 23% in theexamined target organs (liver and kidneys). The AUC(0-3 h) shows 10-13%bioavailability. When formulated as a microemulsion (Formulation 4c), inthe absence of any penetration enhancers, the absolute bioavailabilitywas surprisingly found to be 19-29% in the target organs (liver andkidneys). Formulation 4c provided an AUC(0-3 h) showing about 13%bioavailability that is comparable to the bioavailability seen withFormulations 4a and 4b that used penetration enhancers in solution.However, it should be noted that the AUC(0-3 h) comparison tends tounderestimate the bioavailability, since the blood concentration fromthe intestinal instillation is much higher than that from i.v. injectionat 3 hours after dosing.

TABLE 8 Percent Absolute Bioavailability (% i.v.) of ISIS 2302 FollowingIntrajejunum Instillation in Rats Formulation AUC (0-3 h) No. (Dose)Liver¹ Kidney¹ (ug × h/mL)² Formulation 4a 17.4 17.8 10.7 (80 mg/kg)Formulation 4b 8.8 23.0 13.5 (80 mg/kg) Formulation 4c 19.8 29.1 13.6(48 mg/kg) ¹According to the CGE analysis - total oligonucleotide.²According to analysis by radioactivity. AUC(0-3 h) was calculated forall in vivo instillation studies because the results from radioactivitymeasurements are comparable to those from HPLC analyses for the first 3hour plasma samples.

Example 5 Preparation of Microemulsion Formulations

In order to evaluate the bioavailability of oligonucleotidemicroemulsions the following microemulsion formulations of ISIS 2302were prepared (Table 9)

TABLE 9 Microemulsion Formulations 5a and 5b Formulation ISIS 2302 No.Concentration Components 5a 12 mg/ml Captex 355 3 parts Capmul MCM 1part Tween 80 0.5 part 5b  4 mg/ml Captex 355 3 parts Capmul MCM 1 partTween 80 0.04 part

Formulation 5a:

A microemulsion of ISIS 2302 was prepared essentially according to theprocedures of Panayiotis (Pharm. Res., 1984, 11:1385). An aliquot of 0.6ml of ISIS 2302 stock solution (200 mg/ml) was transferred to a 30 mlbeaker containing 1.0 ml of Tween 80 (Sigma Chemical Company St. Louis,Mo.). Next, a mixture of 6.3 ml of Captex 355 (Abitec Corp., Janesville,Wis.) and 2.1 ml of Capmul MCM (Abitec Corp., Janesville, Wis.) wasadded to the beaker. The resultant solution was stirred until a clearsolution was formed.

Formulation 5b:

A water-in-oil microemulsion of ISIS 2302 was prepared essentially byadding the oil phase to the aqueous phase with adequate mixing. Theaqueous phase was prepared by mixing 1 ml of a 100 mg/ml solution ofISIS 2302 and 1 ml of Tween 80 (Sigma Chemical Company St. Louis, Mo.).The oil phase was prepared by mixing 3 parts of Captex 355 (AbitecCorp., Janesville, Wis.) and 1 part of Capmul MCM (Abitec Corp.,Janesville, Wis.). The oil phase was added to the aqueous phase withadequate stirring until the resultant mixture was a clear solution.

Example 6 Preparation of Water-in-Oil (w/o) Cream Formulations

In order to evaluate the bioavailability of oligonucleotide emulsionsthe water-in-oil cream formulations of ISIS 2302 described in Table 10were prepared as follows.

Formulation 6a1:

A water-in-oil cream formulation of ISIS 2302 was prepared by firstpreparing the two phases. A 2 ml aliquot of the ISIS 2302 stock solution(100 mg/ml) was mixed with 2 ml water in a 10 ml beaker and warmed to70° C. A mixture of 1 gram of Grill 3 (Croda, U.S.), 3 ml Captex 355(Abitec Corp., Janesville, Wis.) and 3 ml of Labrasol (Gattefosse,France) was prepared in a 30 ml beaker and this mixture was also warmedto 70° C. The aqueous solution of oligonucleotide was then poured slowlyinto the oil phase with vigorous mixing. Upon cooling to ambienttemperature the desired water-in-oil cream formulation ofoligonucleotide (−20 mg/mL) was obtained.

Formulation 6a2:

A water-in-oil cream formulation of ISIS 2302 was prepared by firstpreparing the two phases. A 1.5 ml aliquot of the ISIS 2302 stocksolution (200 mg/ml) was transferred to a 10 ml beaker and warmed to 70°C. In a 30 ml beaker were placed 0.5 gram of Grill 3 (Croda, U.S.), 1.5ml Captex 355 (Abitec Corp., Janesville, Wis.), and 1.5 ml of Labrasol(Gattefosse, France) and this mixture also warmed to 70° C. The aqueoussolution of oligonucleotide was then poured slowly into the oil phasewith vigorous mixing. Upon cooling to ambient temperature the desiredwater-in-oil cream formulation oligonucleotide (˜60 mg/mL) was obtained.

TABLE 10 Water-in-Oil Cream Formulations 6a1 (~20 mg/mL): Aqueous Phase2 ml ISIS 2302 solution (100 mg/ml) 2 mL water Oil Phase 1 g. Grill 3(Sorbitan Monostearate) 3 ml Captex 355 3 ml Labrasol 6a2 (~60 mg/mL)Aqueous Phase 1.5 ml ISIS 2302 solution (200 mg/ml) Oil Phase 0.5 g.Grill 3 (Sorbitan Monostearate) 1.5 ml Captex 355 1.5 ml Labrasol

Example 7 Preparation of Oil-in-Water (o/w) Cream

Formulations

In order to evaluate the bioavailability of oligonucleotide emulsionsthe following oil-in-water cream formulations of ISIS 2302 were prepared(Table 11).

TABLE 11 Oil-in-Water Cream Formulation 7a Aqueous Phase 0.5 ml ISIS2302 solution (200 mg/ml) 0.5 ml Tween 80 1.8 ml water Oil Phase 100 mg.Grill 3 (Sorbitan Monostearate) 1 ml Captex 355 1 ml Labrasol

Formulation 7a:

An oil-in-water cream formulation of ISIS 2302 was prepared by firstpreparing the two phases. A 0.5 ml aliquot of the ISIS 2302 stocksolution (200 mg/ml) was mixed with 0.5 ml of Tween 80 (Sigma ChemicalCompany St. Louis, Mo.) and 1.8 ml water in a 30 ml beaker and warmed toabout 70° C. In a 10 ml beaker were placed 100 mg. of Grill 3 (Croda,U.S.), 1 ml Captex 355 (Abitec Corp., Janesville, Wis.), and 1 ml ofLabrasol (Gattefosse, France) and this mixture also warmed to about 70°C. The oil phase was then poured into the aqueous solution ofoligonucleotide with vigorous mixing. Upon cooling to ambienttemperature the desired oil-in-water cream formulation was obtained.

Example 8 In Vivo Evaluation of Emulsion Formulations of ISIS 2302

(A) Following In Vivo Intrajejunum Instillation

In order to determine the ability of emulsion formulations toeffectively deliver oligonucleotide drugs with adequate bioavailabilitythe emulsion formulations of the invention were administered viaintrajejunum instillation and the plasma concentrations of theoligonucleotide and AUC(0-3 h) were measured.

Methods:

Sprague-Dawley rats weighing 250-300 g were used. After overnightfasting, the rats were anesthetized with 5% pentobarbital (50 mg/kg) byintraperitoneal injection. After a midline abdominal incision was made,the small intestine was pulled out and injection site was located (2 cmafter the ligament of Treitz). An aliquot of 0.5 mL drug formulation wasthen injected via a 27 gauge needle. The intestine was put back into thebody carefully. The incision portion was then covered with a wet gauze.300 μL of blood was collected by a 27 gauge needle from the femoral veinat each sampling time point. The rats were sacrificed in a carbondioxide chamber 3 hours after dosing. Plasma samples were analyzed byHPLC.

Formulations:

A 20 mg/ml solution of ISIS 2302 was used as a control formulation.Water-in-oil cream formulations (Formulation 6a1 and 6a2) were used asthe test formulation at two dosage levels—10 mg/rat (6a1) and 30 mg/rat(6a2).

Results:

The results of study are summarized in Table 12. No significant amount(i.e., ˜0%) of ISIS 2302 (SEQ ID NO:1) was absorbed when a controlsolution of the oligonucleotide was used. In contrast, when formulatedas a water-in-oil cream (Formulation 6a1), the blood concentration ofISIS 2302 was found to reach a high of about 34 μg/ml (as determined byHPLC) within 0.5 h of dosing at 10 mg/rat. The concentration of totaloligonucleotides, which includes n-1 oligo and related ISIS 2302metabolites, was observed to be as high as about 36 μg/ml within 0.5 hof dosing. When the rats were dosed at 30 mg/animal (formulation 6a2)the blood concentration of ISIS 2302 was found to reach a high of about64 μg/ml (and total oligonucleotides reached 81 μg/ml) within 0.5 h ofdosing. The AUC(0-3 h) of ISIS 2302 was determined to be 55 ug×h/mL fromthe 10 mg/rat dose and 81 ug×h/mL from the 30 mg/rat dosing.

TABLE 12 Blood Concentrations¹ and AUC(0-3 h) of ISIS 2302 AfterIntrajejunum Instillation in Rats ISIS 2302 Total ISIS 2302 Total OligoAUC AUC Formulation Time Blood Conc. Blood Conc. (0-3 h) (0-3 h)No./Dose (h.) (ug/ml) (ug/ml) (ug · h/ml) (ug · h/ml) 6a1 0.5 33.7335.98 55.24 50.64 1 29.53 29.11 10 mg/rat 2 16.45 15.15 3 0.33 6a2 0.564.11 80.63 81.52 101.19 1 48.49 61.77 30 mg/rat 2 17.67 21.28 3 6.896.70 ¹As determined by HPLC analysis.

(B) Following Rectal Administration

In order to determine the ability of emulsion formulations toeffectively deliver oligonucleotide drugs with adequate bioavailabilitythe emulsion formulations of the invention were administered via therectum and the blood concentrations of the oligonucleotide and AUC(0-3h) were measured.

Methods:

Sprague-Dawley rats weighing 250-300 g were used. After overnightfasting, the rats were anesthetized with 5% pentobarbital (50 mg/kg) byintraperitoneal injection. Test rats were first administered a cleansingenema and then dosed with a sample of the test formulation. A 0.5 mlformulation was applied via a 2 cm length catheter. The rat was liftedup by the bottom in a 15 degree angle to prevent the sample leakageduring the experimental period. 300 μL of blood was collected by a 27gauge needle from the femoral vein at each sampling time point. The ratswere sacrificed in a carbon dioxide chamber 3 hours after dosing. Plasmasamples were analyzed by HPLC.

Formulations:

A 20 mg/ml solution of ISIS 2302 was used as a control formulation.Three different emulsions of 2302 were evaluated as test formulations: awater-in-oil microemulsion (Formulation 5b), a water-in-oil creamformulation (Formulations 6a1 and 6a2) and an oil-in-water creamformulation (Formulation 7a).

Results:

The results of study are summarized in Table 13. No significant amount(L e., ˜0%) of ISIS 2302 (SEQ ID NO:1) was absorbed when a controlsolution of the oligonucleotide was used. In contrast, when theoligonucleotide was administered rectally as a water-in-oilmicroemulsion (Formulation 5b) at 10 mg/rat significant absorption ofthe oligonucleotide occurred as observed from blood levels of about 21μg/mL within 0.5 h and an AUC(0-2 h) of about 28 ug×h/mL.

When formulated as a water-in-oil cream (Formulation 6a1) the plasmaconcentration of ISIS 2302 was found to reach a high of about 34 μg/ml(as determined by HPLC) within 0.5-1.0 h of dosing at 10 mg/rat. Theconcentration of total oligonucleotides, which includes N-1 oligo andrelated ISIS 2302 metabolites was observed to be as high as about 45μg/ml within 0.5-1.0 h of dosing. When the rats were dosed at 30mg/animal (formulation 6a2) the plasma concentration of ISIS 2302 wasfound to reach a high of about 105 μg/ml (and total oligonucleotidesreached 136 μg/ml) within 0.5 h of dosing. The AUC(0-3 h) was determinedto be 64 ug×h/mL from the 10 mg/rat dose and 143 ug×h/mL from the 30mg/rat dosing. A similar increase in delivery of the oligonucleotideinto the blood circulation was observed with the oil-in-water cream(Formulation 7a) when administered rectally at a dose of 10 mg/rat, butthe AUC(0-3 h) was lower than that observed with the water-in-oil cream.

Example 9 Determination of Bioavailability of Oligonucleotides FollowingIntrajejunal and Rectal Administration of Formulations

In order to determine the bioavailability of formulations ofoligonucleotide drugs, two different modes of administration ofoligonucleotide formulated in Formulation 6a1 were studied.

Methods:

Intrajejunal Instillation:

Sprague-Dawley rats weighing 250-300 g were used. After overnightfasting, the rats were anesthetized with 5% pentobarbital (50 mg/kg) byintraperitoneal injection. After a midline abdominal incision was made,the small intestine was pulled out and injection site was located (2 cmafter the ligament of Treitz). An aliquot of 0.5 mL drug formulation wasthen injected via a 27 gauge needle. The intestine was put back into thebody carefully.

TABLE 13 Plasma Concentrations¹ and AUC of ISIS 2302 After RectalAdministration of Formulations to Rats ISIS 2302 Total Oligo Formulationplasma Plasma ISIS 2302 Total No. Time Conc. Conc. AUC AUC & Dose (h.)(μg/ml) (μg/ml) (μg · h/ml) (μg · h/ml) 5b 0.5 21.31 29.00 27.95² 40.85²1 19.00 25.05 10 mg/rat 2 5.71 11.19 3 N/A N/A 6a1 0.5 34.37 45.0764.31³ 84.52³ 1 33.93 44.69 10 mg/rat 2 20.84 27.42 3 10.26 13.37 6a20.5 105.84 136.49 115.85³ 143.29³ 1 71.64 98.49 30 mg/rat 2 20.29 15.473 4.28 5.54 7a 0.5 33.24 43.42 34.96³ 46.03³ 1 21.09 27.42 10 mg/rat 24.87 6.74 3 3.61 4.89 ¹As determined by HPLC analysis. ²AUC (0-2 h).³AUC (0-3 h).

Rectal Administration:

Following a period of overnight fasting, the rats were anesthetized with5% pentobarbital (50 mg/kg). Test rats were first administered acleansing enema and then dosed with a sample of the test formulation.The enema formulation was applied via a 2 cm length catheter. The bottompart of rat was lifted up in a 15 degree angle to hold the solution.

In order to assess bioavailability of oligonucleotide, samples wereprocessed and the amount of oligonucleotide present was assessed by HPLCanalyses.

Results:

The absolute bioavailability of ISIS 2302 (SEQ ID NO: 1) was determinedfollowing intrajejunal instillation in five Sprague-Daley rats andfollowing rectal administration in seven rats. The results are shown inTable 14.

TABLE 14 Bioavailability of ISIS 2302 Following Intrajejunal and RectalAdministration in Rats Route of Administration Absolute BioavailabilityIntrajejunal 20.3% (n = 5) Rectal 24.5% (n = 7)

Example 10 Preparation of Emulsion Formulations Containing PenetrationEnhancers

Various fatty acids, their salts and their derivatives act aspenetration enhancers. These include, for example, oleic acid, a.k.a.cis-9-octadecenoic acid (or a pharmaceutically acceptable salt thereof,e.g., sodium oleate or potassium oleate); caprylic acid, a.k.a.n-octanoic acid (caprylate); capric acid, a.k.a. n-decanoic acid(caprate); lauric acid (laurate); acylcarnitines; acylcholines; andmono- and di-glycerides (Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92).

Various natural bile salts, and their synthetic derivatives act aspenetration enhancers. The physiological roles of bile include thefacilitation of dispersion and absorption of lipids and fat-solublevitamins (Brunton, Chapter 38 In: Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 9th Ed., Goodman et al., eds., McGraw-Hill, NewYork, N.Y., 1996, pages 934-935). Bile salt derived penetrationenhancers include, for example, cholic acid, cholalic acid or3a,7a,12a-trihydroxy-5b-cholan-24-oic acid (or its pharmaceuticallyacceptable sodium salt); deoxycholic acid, desoxycholic acid,5b-cholan-24-oic acid-3a,12a-diol, 7-deoxycholic acid or3a,12a-dihydroxy-5b-cholan-24-oic acid (sodium deoxycholate);glycocholic acid, (N-[3a,7a,12a-trihydroxy-24-oxocholan-24-yl]glycine or3a,7a,12a-trihydroxy-5b-cholan-24-oic acid N-[carboxymethyl]amide orsodium glycocholate); glycodeoxycholic acid, (5b-cholan-24-oic acidN-[carboxymethyl]amide-3a,12a-diol), 3a,12a-dihydroxy-5b-cholan-24-oicacid N-[carboxymethyl]amide,N-[3a,12a-dihydroxy-24-oxocholan-24-yl]glycine or glycodesoxycholic acid(sodium glycodeoxycholate); taurocholic acid, (5b-cholan-24-oic acidN-[2-sulfoethyl]amide-3a,7a,12a-triol),3a,7a,12a-trihydroxy-5b-cholan-24-oic acid N-[2-sulfoethyl]amide or2-[(3a,7a,12a-trihydroxy-24-oxo-5b-cholan-24-yl)amino]ethanesulfonicacid (sodium taurocholate); taurodeoxycholic acid,(3a,12a-dihydroxy-5b-cholan-2-oic acid N[2-sulfoethyl]amide or2-[(3a,12a-dihydroxy-24-oxo-5b-cholan-24-yl)-amino]ethanesulfonic acid,or sodium taurodeoxycholate, or sodium taurodesoxycholate);chenodeoxycholic acid (chenodiol, chenodesoxycholic acid, 5b-cholanicacid-3a,7a-diol, 3a,7a-dihydroxy-5b-cholanic acid, or sodiumchenodeoxycholate, or CDCA); ursodeoxycholic acid, (5b-cholan-24-oicacid-3a,7b-diol, 7b-hydroxylithocholic acid or3a,7b-dihydroxy-5b-cholan-24-oic acid, or UDCA); sodiumtaurodihydro-fusidate (STDHF); and sodium glycodihydrofusidate (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783). Commercial sources of the penetration enhancers are listed inTable 15.

TABLE 15 Sources of Penetration Enhancers Compound Name AbbreviationSupplier A. FATTY ACIDS AND DERIVATIVES Capric acid, Na salt caprateSigma^(H) Lauric acid, Na salt laurate Sigma B. BILE SALTS ANDDERIVATIVES Cholic acid, Na salt CA Sigma Glycholic acid, Na salt GCASigma Glycodeoxycholic acid, Na Salt GDCA Sigma Taurocholic acid, Nasalt TCA Sigma Taurodeoxycholic acid, Na salt TDCA SigmaChenodeoxycholic acid, Na salt CDCA Sigma Ursodeoxycholic acid UDCAAldrich^(I) ^(H)Sigma, Sigma Chemical Company, St. Louis, MO.^(I)Aldrich, Aldrich Chemical Company, Milwaukee, WI.

In order to evaluate the ability of various penetration enhancers toenhance the oral delivery and/or mucosal penetration ofoligonucleotides, and the bioavailability of oligonucleotides thefollowing formulations were prepared (Table 16). Formulations 9a and 9cwere prepared as emulsions of ISIS 2302 containing a combination offatty acid and bile salt penetration enhancers. Formulations 9b and 9dwere prepared such that the concentration of ISIS 2302 and thepenetration enhancers remained the same as in Formulations 9a and 9c,respectively. However, formulations 9b and 9d are merely solutionpreparations and serve as comparison points for the emulsionformulations.

TABLE 16 ISIS 2302 Formulations Containing Penetration Enhancers (PE's)9a-9d Formulation Oil Phase Aqueous Phase 9a Captex 355 1.25 ml ISIS2302 0.5 ml Labrasol 1.25 ml Mix. Of PE's 1.0 ml Grill 3  0.5 g. Water0.5 ml The aqueous phase contains 2% CDCA, 4% Sodium Laurate, and 4%Sodium Caprate 9b Solution of ISIS 2302 (20 mg/ml) containing 2% CDCA,4% Sodium Laurate, and 4% Sodium Caprate 9c Captex 355 1.25 ml ISIS 23020.5 ml Labrasol 1.25 ml Mix. Of PE's 1.0 ml Grill 3  0.5 g. Water 0.5 mlThe aqueous phase contains 2% UDCA, 4% Sodium Laurate, and 4% SodiumCaprate 9d Solution of ISIS 2302 (20 mg/ml) containing 2% UDCA, 4%Sodium Laurate, and 4% Sodium Caprate Note: ISIS 2302 Stock Solutionused was 200 mg/ml.

Formulation 9a:

An emulsion formulation of ISIS 2302 was prepared by first preparing thetwo phases. A 0.5 ml aliquot of the ISIS 2302 stock solution (200 mg/ml)was mixed with 1.0 ml of the mixture of penetration enhancers(chenodeoxycholic acid sodium salt, sodium laurate and sodium caprate)and 0.5 ml water, and warmed to about 70° C. (aqueous phase). A separatemixture of 500 mg. of Grill 3 (Croda International Plc., East Yorkshire,U.K.), 1.25 ml Captex 355 (Abitec Corp., Janesville, Wis.), and 1.25 mlof Labrasol (Gattefosse Corp., Westwood, N.J.) was also prepared andwarmed to about 70° C. (oil phase). The aqueous phase was thentransferred to the oil phase with vigorous mixing to afford the desiredemulsion concentration of 20 mg/ml ISIS 2302. The aqueous phase ofemulsion contained 2% CDCA, 4% Sodium laurate, and 4% Sodium caprate.

Formulation 9b:

Aliquots of the stock solution of ISIS 2302 (200 mg/ml) and mixture ofpenetration enhancers were mixed to afford a solution formulationcomprising ISIS 2302 at a concentration of 20 mg/ml and a finalconcentration of 2% CDCA, 4% Sodium laurate and 4% Sodium caprate.

Formulation 9c:

An emulsion formulation of ISIS 2302 was prepared by first preparing thetwo phases. A 0.5 ml aliquot of the ISIS 2302 stock solution (200 mg/ml)was mixed with 1.0 ml of the mixture of penetration enhancers(ursodeoxycholic acid sodium salt, sodium laurate and sodium caprate)and 0.5 ml water, and warmed to about 70° C. A separate mixture of 500mg. of Grill 3 (Croda, U.S.), 1.25 ml Captex 355 (Abitec Corp.,Janesville, Wis.), and 1.25 ml of Labrasol (Gattefosse, France) was alsoprepared and warmed to about 70° C. The aqueous phase was thentransferred to the oil phase with vigorous mixing to afford the desiredemulsion concentration of 20 mg/ml ISIS 2302. The aqueous phase ofemulsion contained 2% UDCA, 4% Sodium laurate, and 4% Sodium caprate.

Formulation 9d:

Aliquots of the stock solution of ISIS 2302 (200 mg/ml) and mixture ofpenetration enhancers were mixed to afford a solution formulationcomprising ISIS 2302 at a concentration of 20 mg/ml and a finalconcentration of 2% UDCA, 4% Sodium laurate, and 4% Sodium caprate.

Example 11 Evaluation of Emulsion Formulations Containing PenetrationEnhancers

In order to determine the ability of penetration enhancers to improvethe absorption and delivery of oligonucleotide drugs the emulsionformulations of Example 9 were administered via intrajejunuminstillation and the blood concentrations of the oligonucleotide andAUC(0-3 h) were measured.

Methods:

Sprague-Dawley rats weighing 250-300 g were used. After overnightfasting, the rats were anesthetized with 5% pentobarbital (50 mg/kg) byintraperitoneal injection. After a midline abdominal incision was made,the small intestine was pulled out and injection site was located (2 cmafter the ligament of Treitz). An aliquot of 0.5 mL drug formulation wasthen injected via a 27 gauge needle. The intestine was put back into thebody carefully. The incision portion was covered with a wet gauze. 300μL of blood was collected by a 27 gauge needle from the femoral vein ateach sampling time point.

Formulations:

Two emulsions (Formulations 9a and 9c) were evaluated at a dosage levelof 10 mg/rat. The delivery of ISIS 2302 using these emulsions(Formulations 9a and 9c) formulated with a combination of penetrationenhancers was compared to the performance of solutions (Formulations 9band 9d, respectively) of ISIS 2302 that were formulated with the samecombination and concentration of penetration enhancers.

Results:

The results of study are summarized in Table 17. When a control solutionof ISIS 2302 (SEQ ID NO:1) was administered no significant amount ofoligonucleotides was found to be absorbed. In contrast, when ISIS 2302was formulated as a solution that contained a mixture of fatty acid andbile salts (Formulations 9b and 9d) a significant amount ofoligonucleotide was found to be absorbed and present in the systemiccirculation. The concentrations of oligonucleotide in plasma were foundto be in the 42-73 μg/ml concentration range 30 mins afteradministration of Formulation 9b (at a dose of 10 mg/rat) which contains2% CDCA. At the same time point 33-87 μg/ml of oligonucleotide wasdelivered into the blood circulation when Formulation 9d containing 2%UDCA was administered (at a dose of 10 mg/rat). The AUC(0-3 h) wasobserved to be in the 48-58 ug×h/mL range from Formulation 9b and the30-101 ug×h/mL range from administration of Formulation 9d.

Further increases in the amount of oligonucleotides delivered intosystemic circulation were observed when the emulsions containing 2% bilesalt together with 4% each of sodium laurate and sodium caprate wereevaluated. Thus, Formulation 9a which incorporates the bile salt CDCAafforded 34-52 μg/ml concentrations of total oligonucleotide in theplasma within 30 minutes of administration and an AUC(0-3 h) in the63-151 ug×h/mL range. Likewise, Formulation 9c which incorporates thebile salt UDCA afforded 54-79 μg/ml concentrations of totaloligonucleotide in the plasma within 30 minutes of administration and anAUC(0-3 h) in the 84-127 μg·h/ml range.

TABLE 17 Plasma Concentrations¹ and AUC(0-3 h) of ISIS 2302 AfterIntrajejunum Instillation in Rats Conc. Conc. Total Animal (mg/ml) TimeTotal Animal (mg/ml) Formulation #1 #2 #3 (h.) Formulation #1 #2 #3 9a52.2 34.0 96.04 0.5 9b 42.8 72.9 45.7 50.1 33.2 89.62 1 26.1 15.0 29.919.7 17.6 41.67 2 10.4 15.4 7.9 7.7 14.6 12.89 3 4.3 7.4 4.7 80.6 62.54151.25 AUC (0-3 h) 48.13 57.7 49.8 9c 54.8 53.7 78.6 0.5 9d 33.3. 87.353.4 56.6 84.6 1 17.1 63.0 18.0 17.4 32.0 2 4.2 18.6 10.6 5.1 5.0 3 0.65.1 83.9 82.54 127.44 AUC (0-3 h) 30.57 101.15 ¹As determined by HPLCanalysis.

Example 11 Comparison of ISIS 2302 Bioavailability from FormulationsAdministered by Intrejenunal Instillation

In order to determine the effectiveness of formulations the absolutebioavailability of oligonucleotide was assessed following intrajejunalinstillation.

Methods:

Sprague-Dawley rats weighing 250-300 g were used. After overnightfasting, the rats were anesthetized with 5% pentobarbital (50 mg/kg) byintraperitoneal injection. After a midline abdominal incision was made,the small intestine was pulled out and injection site was located (2 cmafter the ligament of Treitz). An aliquot of 0.5 mL drug formulation wasthen injected via a 27 gauge needle. The intestine was put back into thebody carefully. The incision portion was covered with a wet gauze. 300μL of blood was collected by a 27 gauge needle from the femoral vein ateach sampling time point. The rats were sacrificed in a carbon dioxidechamber 3 hours after dosing. Plasma samples were analyzed by HPLC.

Formulations:

Five formulations were evaluated. Two solution formulations wereprepared. Formulation 9b was prepared by dissolving ISIS 2302 and acombination of CDCA and fatty acid penetration enhancers to the desiredconcentrations. Formulation 9d was prepared by dissolving ISIS 2302 anda combination of UDCA and fatty acid penetration enhancers to thedesired concentrations.

Formulation 6a1 was prepared as an emulsion of ISIS 2302 in a mixture oflabrasol, captex and Grill 3. Formulation 9a was also prepared as anemulsion that is similar to Formulation 6a1 with one difference in thatCDCA and fatty acid penetration enhancers were incorporated into aqueousphase of Formulation 9a at the same concentrations as were present inFormulation 9b. Formulation 9c was likewise prepared as an emulsion thatis similar to Formulation 6a1 with one difference in that UDCA and fattyacid penetration enhancers were incorporated into the aqueous phase ofFormulation 9c at the same concentrations as were present in Formulation9d.

All five formulations contained ISIS 2302 at a final concentration of 20mg/ml.

Results:

The results of absolute bioavailability of ISIS 2302 as determined inthis study are summarized in Table 18.

TABLE 18 Absolute Bioavailability of ISIS 2302 Following IntrajejunalInstillation in Rats Absolute Formulation Composition Bioavailability 9bISIS 2302 + CDCA + Fatty acids 14.6% (n = 5) solution 9a ISIS 2302 +CDCA + Fatty acids + 27.7% (n = 3) emulsion Labrasol + Captex + Grill 39d ISIS 2302 + UDCA + Fatty acids 12.4% (n = 2) solution 9c ISIS 2302 +UDCA + Fatty acids + 27.7% (n = 3) emulsion Labrasol + Captex + Grill 36a1 ISIS 2302 + 20.3% (n = 5) emulsion Labrasol + Captex + Grill 3

When a control solution of ISIS 2302 (SEQ ID NO:1) was administered, nosignificant amount of oligonucleotides was found to be absorbed. Incontrast, when ISIS 2302 was formulated as a solution that contained amixture of fatty acids and a bile salt (Formulations 9b and 9d), asignificant amount of oligonucleotide was found to be absorbed andbioavailable in the systemic circulation. The absolute bioavailabilityof ISIS 2302 was found to be 14.6% from Formulation 9b (containing amixture of CDCA and fatty acid penetration enhancers) and 12.4% fromFormulation 9d (containing a mixture of UDCA and fatty acid penetrationenhancers). The simple emulsion, Formulation 6a1, that is devoid of anypenetration enhancers was also effective in making a significant portionof the ISIS 2302 oligonucleotide bioavailable (absolute bioavailabilityof 20.4%).

When formulated as emulsions that contained a combination of penetrationenhancers, the bioavailability of ISIS 2302 was found to increase evenfurther. Formulation 9a, which is an emulsion containing a combinationof CDCA and fatty acid penetration enhancers, afforded an absolutebioavailability of ISIS 2302 to the tune of 27.7%. A similarbioavailability of the oligonucleotide was also found when emulsionFormulation 9c containing UDCA instead of CDCA, as in Formulation 9a,was evaluated.

Example 13 Preparation of Enema Formulations

To evaluate the delivery and mucosal penetration of oligonucleotidesinto the colon following rectal delivery, the following formulationswere prepared (Table 19). Solution and emulsion formulations of ISIS2302 (SEQ ID NO: 1) were prepared. Additives used in the formulationsincluded saline, hydroxypropyl methyl cellulose (HPMC), carrageenan,Vitamin E a-tocopheryl polyethyelene glycol 1000 succinate (TPGS), Tween80 and sorbitol.

TABLE 19 ISIS 2302 Formulations 12a-12f Formulation Composition 12a ISIS2302 in Saline 12b ISIS 2302 + 1.5% Hydroxypropyl Methyl Cellulose(HPMC) 12c ISIS 2302 + 1.0% Carrageenan + 2.5% Vitamin E a-TocopherylPolyethylene Glycol 1000 Succinate (TPGS) (Source: Eastman ChemicalCompany, NY) 12d ISIS 2302 in a w/o emulsion 12e ISIS 2302 + 0.5% Tween80 + 0.75% HPMC 12f ISIS 2302 + 5% Sorbitol + 0.63% HPMC 12g ISIS 2302in water

Formulation 12a:

A solution of ISIS 2302 was prepared by mixing 5 ml ISIS 2302 stocksolution (100 mg/mL) with 95 ml sterile saline to have a finalconcentration of 5 mg/ml.

Formulation 12b:

The solution was prepared by mixing 7.5 ml ISIS 2302 stock solution (100mg/mL) with 142.5 ml hydroxypropyl methyl cellulose solution (HPMC) tohave final concentrations of ISIS 2302 5 mg/ml and HPMC 15 mg/ml (1.5%).The HPMC solution was prepared by dissolving 2.25 g HPMC in 30 ml of 80°C. water and Q.S. to 142.5 mL with cold water.

Formulation 12c:

The solution was prepared by mixing 7.5 ml ISIS 2302 stock solution (100mg/ml) with 142.5 ml carrageenan/Vitamine E TPGS solution to have afinal concentration of ISIS 2302 5 mg/ml, carrageenan 10 mg/ml (1%), andVitamine E TPGS 25 mg/ml (2.5%). The carrageenan/Vitamine E TPGSsolution was prepared by dissolving 1.5 g carrageenan and 3.75 g VitaminE TPGS in 142.5 ml water. The solution was then heated to 60° C. to forma gel and cooled down to room temperature before the addition of ISIS2302 stock solution.

Formulation 12d:

A water-in-oil emulsion (w/o) of ISIS 2302 was prepared following thegeneral methods in Examples 5 and 6. The emulsion containing 5 mg/ml ofISIS 2302.

Formulation 12e:

The solution was prepared by mixing 6.0 ml ISIS 2302 stock solution (100mg/ml) with 0.6 ml tween 80, and 113.4 ml HPMC solution to have finalconcentrations of ISIS 2302 5 mg/ml, tween 80 50 μl/ml (0.5%), and HPMC7.5 mg/ml (0.75%). The HPMC solution was prepared by dissolving 0.9 gHPMC in 60 ml of 80° C. water and Q.S. to 113.4 ml with cool water.

Formulation 12f:

The solution was prepared by mixing 6.0 ml ISIS 2302 stock solution (100mg/ml) with 6 g sorbitol, and 108 ml HPMC solution to have finalconcentrations of ISIS 2302 5 mg/ml, sorbitol 50 mg/ml (5%), and HPMC6.3 mg/ml (0.63%). The HPMC solution was prepared by dissolving 0.75 gHPMC in 50 ml of 80° C. water and Q.S. to 108 ml with cool water.

Formulation 12g:

A solution of ISIS 2302 was prepared by mixing 5 ml ISIS 2302 stocksolution (100 mg/ml) with 95 ml water to have a final concentration of 5mg/ml

Example 14 Evaluation of Enema Formulations for Local Delivery ofOligonucleotide

Formulations of oligonucleotide were evaluated via rectal administrationas enemas to laboratory beagle dogs.

Methods:

Following a period of overnight fasting, test dogs were firstadministered a cleansing enema and then dosed with a sample of the testformulation. The enema formulation was applied via a Foley catheter andhold for a period of 1 h. In order to assess colonic tissue delivery anduptake of oligonucleotide, colon tissue biopsies were performed on thetest animal, 3 h and 24 h after dosing. Tissue samples were processedand the amount of oligonucleotide present in the tissue assessed bycapillary gel electrophoresis (CGE) and immunohistochemical (1HC)analyses.

Results:

Seven formulations of ISIS 2302 (SEQ ID NO: 1) as prepared in Example 12(Formulations 12a-12g) were administered to dogs via rectal enemas andthe local distribution of ISIS 2302 in colonic tissue was determined byCGE and IHC at 3 h and 24 h following dosing. The results are shown inTable 20.

TABLE 20 Local Colonic Tissue Distribution of ISIS 2302 Following RectalEnema in Dog IHC CGE (μg/g) Formulation 3 h 24 h 3 h 24 h 12a ++++ − 782± 664 NA 12b ++++ − 660 ± 440 6.8 ± 5.0 12c ++++ − 558 ± 212 2.5 ± 1.412d ++++ − 224 ± 78  1.2 ± 0.7 12e ++++ − 621 ± 368 6.0 ± 5.9 12f ++++ −417 ± 127 1.3 ± 0.5 12g ++++ − 143 NA Note: “++++” indicates strongstaining in IHC using a primary antibody to ISIS 2302; “−” indicates nosignificant staining compared to background levels.

Surprisingly, effective local colonic delivery of oligonucleotide wasseen for a variety of formulations. Even oligonucleotide that was merelysuspended in sterile saline (Formulation 12a) or water (Formulation12g). The presence of HPMC, a protective colloid that is useful as ageneral dispersing and thickening excipient, did not hinder thelocalized delivery of oligonucleotide (Formulation 12b), nor did otheradditives, such as Carrageenan and TPGS (12c) Tween 80 (12e) or Sorbitol(12f).

The present disclosure thus provides for localized delivery ofoligonucleotides and other small nucleic acids to the lower portion ofthe G.I. tract. Such delivery can be via means of an enema using asolution comprising an effective concentration of oligonucleotide.Alternatively, suppositories comprising oligonucleotides suspended in anagent that disperses its contents when exposed to the physical and/orchemical conditions of the colon. In addition to HMPC, a preferreddispersing agent for localized colconic delivery of oligonucleotides iscocoa butter.

Immunohistochemical (IHC) analyses were used to confirm and extend theseresults by determining the histopathology and cellular localization ofrectally administered oligonucleotides in dogs. Biopsy samples weretaken 10 to 20 centimeters from (i.e., approximately 18, 20, 21 and 22cm from) the dorsal side of the colon and evaluated for the distributionof phosphorothioate oligodeoxynucleotide (P═S ODN) ISIS-2302 three hoursafter rectal administration. The biopsy samples were fixed in 10%neutral buffered formalin for 24 hours and transferred to 70% forstorage. The tissues were embedded in paraffin and sectioned at 5 μm forimmunohistochemical detection of P═S ODN. The affinity purified antibodyused for this work, 2E1-B5, is a mouse IgG1 (Berkeley Antibody Company,Richmond, Calif.) which specifically recognizes P═S ODN.

Tissues were deparaffinized and pre-treated with proteinase K (DakoCorp., Carpenteria, Calif.) for 10 minutes at room temperature prior toincubation in the primary antibody. The antibody was detected withdonkey anti mouse f(ab′)2 IgG conjugated to horseradish peroxidase(Jackson Laboratories, West Grove, Pa.) and diaminobenzidene (DAB, DakoCorp.) was used as a substrate. All slides were stained on the Dakoautomated immuno-stainer.

Staining of the P═S ODN is seen in the nucleus of the surface epitheliallayer at the tips of the colonic villi in all of the biopsies. Thestaining is strongest in the 20-22 cm samples and some staining is seenat the luminal surface of the epithelium, which is most likelyassociated with mucinous material in the colon.

Example 15 Bioavailability of Oligonucleotide Tablet Formulations: ISIS2302 and ISIS 15839

In order to evaluate the potential for delivering oligonucleotides viavarious oral dosage forms, the following experiments were carried out.

A. Composition of and Preparation of Oral Dosage Formulations

The following oral dosage formulations of oligonucleotides were preparedas follows.

Oral Dosage Formulation a:

ISIS 2302 with penetration enhancers (CDCA, SC, SL) and excipientprecirol.

Oligonucleotide (ISIS 2302) was passed through a 60 mesh screen, 12.4 gof which was then mixed with 10 g sodium chenodeoxycholate (CDCA), 20 gof sodium caprate (SC), 20 g sodium laurate (SL) and 47.5 g precirol (WL2155 ATO, prescreened on 60 mesh). The powder was then placed in aplastic bag, mixed thoroughly and then sifted through a 20 mesh screen.Powder blend was then compressed into tablets at slight weight overageusing round flat-faced tooling. The resulting 1100″ 50 mg tabletscontained 124 mg oligonucleotide (as is by weight), 100 mg CDCA, 200 mgSL, 200 mg SC, and 476 mg precirol. Resultant tablets may be used as is(core tablets) or may be enteric film coated as described below under“Enteric Coating”.

Oral Dosage Formulation b:

ISIS 2302 with penetration enhancers (CDCA, SC, SL) and excipient (PEG).

Oligonucleotide (ISIS 2302) was passed through a 60 mesh screen and 9.3g of which was mixed with 7.5 g sodium chenodeoxycholate (CDCA), 15 g ofsodium caprate (SC), 15 g sodium laurate (SL) and 35.7 gpolyethyleneglycol (20,000 mw PEG, prescreened 20 mesh). The powder wasthen placed in a plastic bag, mixed thoroughly and sifted through 20mesh screen. Powder blend was then compressed into tablets at slightweight overage using round flat-faced tooling. The resulting 1100″ 50 mgtablets contained 124 mg oligonucleotide (as is by weight), 100 mg CDCA,200 mg SL, 200 mg SC, and 476 mg PEG. Resultant tablets may be used asis (core tablets) or may be enteric film coated as described below under“Enteric Coating”.

Oral Dosage Formulation c:

ISIS 15839 with penetration enhancers (CDCA, SC, SL) and excipient(PEG).

ISIS 15839 is a phosphorothioate isosequence “hemimer” derivative ofISIS 2302 having the structure 5′-GCC-CAA-GCT-GGC-ATC-CGT-CA-3′ (SEQ IDNO:1), wherein emboldened “C” residues have 5-methylcytosine (m5c) basesand wherein the emboldened, double-underlined residues further comprisea 2′-methoxyethoxy modification (other residues are 2′-deoxy). ISIS15839 is described in co-pending U.S. patent application Ser. No.09/062,416, filed Apr. 17, 1998, hereby incorporated by reference.

ISIS 15839 was passed through a 60 mesh screen and 2.323 g of which wasmixed with 2.0 g sodium chenodeoxycholate (CDCA), 4.0 g of sodiumcaprate (SC), 4.0 g sodium laurate (SL) and 9.523 g polyethyleneglycol(20,000 mw PEG, prescreened 20 mesh). The powder was then sifted througha 20 mesh screen and placed in a plastic bag and mixed thoroughly.Powder blend was then compressed into tablets at slight weight overageusing 12 mm round tooling. The resulting 728.4″ 10 mg tablets contained77.44 mg oligonucleotide, 66.67 mg CDCA, 133.4 mg SL, 133.4 mg SC, and317.5 mg PEG. Resultant tablets may be used as is (core tablets) may beenteric film coated as described below under “Enteric Coating”.

Oral Dosage Formulation d:

ISIS 2302 with penetration enhancers (CDCA, SC, SL) without excipient.

Oligonucleotide ISIS 2302 was passed through a 60 mesh screen and 3.72 gof which was mixed with 3 g sodium chenodeoxycholate (CDCA), 6 g ofsodium caprate (SC) and 6 g sodium laurate (SL). The powder was siftedthrough a 20 mesh screen and placed in plastic bag and mixed thoroughly.This powder blend was then compressed into tablets at slight weightoverage using 12 mm diameter tooling. The resulting 624″ 10 mg tabletscontained 124 mg oligonucleotide (as is by weight), 100 mg CDCA, 200 mgSL and 200 mg SC. Resultant tablets may be used as is (core tablets) ormay be enteric film coated as described below under “Enteric Coating”.

Enteric Coating (EC)

A cellulose acetate phthalate (CAP) enteric coating solution wasprepared by slowly adding 7.0 g of CAP powder to 90.0 g stirred acetone.Before dissolving, 3.0 g diethyl phthalate was added and the solutioncovered with aluminum foil and continued to stir until dissolution wascomplete after approximately 30 minutes.

Core tablets were coated by hand by dipping into a stirred CAP solutionusing vacuum tubing to hold the tablet. As the coating dried the tabletswere inverted and redipped to effect completion of a single coat overentire surface. This method may be repeated to impart an adequateenteric film coverage of 2 to 5% weight gain and, depending on tabletsize and configuration, to allow for a uniform coat thickness andperformance quality.

B. Evaluation of Oral Dosage Formulations

The tablet formulations were evaluated according to the followingmethods.

In Vitro:

In order to evaluate the integrity of the enteric film coat, tabletswere placed in 500 mL aqueous 0.1 N HCL acid solution (pH 1.5) using USPmethod II (paddles) at 150 rpm and 37° C. for up to 1 hour. Filteredsamples were periodically taken and analyzed for presence ofoligonucleotide as described below. Absence of oligonucleotide andvisual inspection verified enteric coat integrity. Tablets were thenplaced into 500 mL of 0.2 M phosphate buffer solution, pH 6.5, toevaluate the rate at which oligonucleotides were released out of thetablets. Dissolution was monitored at regular time intervals byanalyzing sample filtrate using UV light at 260 nm. This analysis wassuitable for formulations devoid of interfering components (i.e.,dissolved excipients capable of absorbing at 260 nm). Alternatively,samples may be analyzed by any of various separation methods (e.g.,HPLC).

In Vivo:

In order to measure the bioavailability of oligonucleotides from tabletformulations, tablets were administered orally (p.o.) to healthy beagledogs of ˜12 kg average weight at an approximate dose of 15 mg/kg. Bloodsamples were taken at regular time intervals and plasma harvested thensubsequently analyzed for presence of oligonucleotide by either highpressure liquid chromatography (HPLC) for screening purposes orcapillary gel electrophoresis (CGE) for purposes of confirmation and/orquantitation. Baseline pharmacokinetic intravenous (i.v.) data wereobtained by administration of sterile drug solution (2 mg/kg) by slowi.v. push via antecubital vein followed by phlebotomies and analysis asdescribed above.

Percent bioavailability was calculated from the resulting data accordingto the following formula% Bioavailability=(AUC_(po) /D _(o))/(AUC_(iv) /D _(o))×100%,wherein AUC_(po) is area under the plasma concentration curve forformulated oligonucleotide tablets administered orally, AUC_(iv) is areaunder the plasma concentration curve for oligonucleotide administered asan i.v. solution (control), and D_(o) is the respective dosages forthese two regimens.Results:

The results of the study are summarized in Table 21. As expected,enteric coated (EC) tablets had longer times of dissolution in acidicconditions (i.e., 4 minutes for ˜50% dissolution for tablets lacking anenteric coating, from 7 to 14 minutes for EC tablets).

As indicated by the C_(max) values, all of the oral dosage formulationstested can result in plasma concentrations of at least 1 ug/mL. Oraldosage formulation a, with or without an enteric coating, had C_(max)values of 2.4 ug/mL and 2.0 mg/mL, respectively. A similar C_(max) valuewas observed with enteric coated tablets of oral dosage formulation c.Oral dosage formulation b had the lowest C_(max) value (1.2 ug/mL) whenno enteric coating was provided but gave the highest C_(max) value (3.3mg/mL) when an enteric coating was used.

The % Bioavailability values generally followed the trend established bythe C_(max) values. For example, Oral dosage formulation b had thelowest % Bioavailability value when no enteric coating was provided butgave the highest % Bioavailability value when an enteric coating wasused. As with C_(max), the remaining oral dosage formulations had %Bioavailability values that are comparable to each other.

TABLE 21 In Vitro Dissolution of, and In Vivo Plasma Levels in DogsResulting from, Oral Dosage Formulations of ISIS 2302 and 15839 OralDosage D₅₀ ¹ C_(max) ² Formulation (min) (μg/mL) % Bioavailability³ acore tablets 4 2.4 2.0 b core tablets 4 1.2 0.9 a with CAP EC⁴ 12 2.02.3 b with CAP EC 14 3.3 2.8 c with CAP EC 7 2.2 2.2 ¹D₅₀ = approximatetime for 50% tablet dissolution. ²C_(max) = maximum oligonucleotideconcentration in plasma. ³Calculated as dose normalized AUC relative toi.v. AUC. ⁴CAP EC = cellulose acetate phthalate (CAP) enteric coating.

Example 16 Use of Other Animal Models to Evaluate Formulations

In order to further evaluate the bioavailability of the formulations ofthe invention, various animal models are used. For example, rectalformulations are tested in rats essentially according to the method ofAungst et al. (Pharm. Res., 1988, 5:305) or in rabbits essentiallyaccording to the methods of Buur et al. (J. Control Rel., 1990, 14:43)and Yamamoto et al. (J. Pharmacol. Exper. Therapeutics, 1992, 263:25).

The formulations of the invention are further evaluated in largeranimals for optimization of the penetration enhancer (PE) systems interms of, e.g., concentration and temporal effects on the absorption ofoligonucleotides (ODN). Dogs will be “ported” with intestinal accesscatheters through which formulated drug formulations (solutions orsuspensions) may be introduced into various areas of the gut. Targetareas include the proximal jejunum and distal ilium or the iliocecaljunction. These respective areas provide for ideal assessment of thesystemic oligonucleotide bioavailability and for local tissue (e.g.,colonic) absorption. This latter objective is assessed on the basis ofboth tissue biopsy drug levels and/or inferred by the presence of drugin the plasma. In addition to ported dogs, naive dogs will be used forthe assessment of formulations given by conventional routes, e.g., oraladministration for oral dosage forms, rectal administration for enema orsuppository formulations, etc. Dogs are dosed at 10 mg/kg ofoligonucleotides, which are appropriately labeled as necessary, andblood samples are collected and evaluated for the presence andconcentration of oligonucleotides. The absolute bioavailability iscalculated and, if necessary, animals are sacrificed and tissue samplesare collected and analyzed.

Those skilled in the art will appreciate that numerous changes andmodifications may be made to the preferred embodiments of the inventionand that such changes and modifications may be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention. Also, it is intended that eachof the patents and patent applications referenced above be incorporatedby reference herein.

A further preferred oligonucleotide modification includes2′-dimethylamino oxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in co-owned U.S. patent application Ser. No.09/016,520, filed on Jan. 30, 1998, the contents of which are hereinincorporated by reference. Other preferred modifications include2′-methoxy (2′-O—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro(2′-F). Similar modifications may also be made at other positions on thesugar group, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. The nucleosides of theoligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar.

Unsubstituted and substituted phosphodiester oligonucleotides arealternately synthesized on an automated DNA synthesizer (AppliedBiosystems model 380B) using standard phosphoramidite chemistry withoxidation by iodine.

Phosphorothioates are synthesized as per the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 hr), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution.

Phosphinate oligonucleotides are prepared as described in U.S. Pat. No.5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, hereby incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively).

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Boranophosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand PO or PS linkages are prepared as described in U.S. Pat. Nos.5,378,825; 5,386,023; 5,489,677; 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Peptide nucleic acids (PNAs) are prepared in accordance with any of thevarious procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5. They may also be prepared in accordance with U.S.Pat. Nos. 5,539,082; 5,700,922, and 5,719,262, herein incorporated byreference.

A further preferred oligonucleotide modification includes2′-dimethylamino oxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in co-owned U.S. patent application Ser. No.09/016,520, filed on Jan. 30, 1998, the contents of which are hereinincorporated by reference. Other preferred modifications include2′-methoxy (2′-O—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro(2′-F). Similar modifications may also be made at other positions on thesugar group, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. The nucleosides of theoligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar.

Unsubstituted and substituted phosphodiester oligonucleotides arealternately synthesized on an automated DNA synthesizer (AppliedBiosystems model 380B) using standard phosphoramidite chemistry withoxidation by iodine.

Phosphorothioates are synthesized as per the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 hr), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution.

Phosphinate oligonucleotides are prepared as described in U.S. Pat. No.5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, hereby incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively).

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Boranophosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand PO or PS linkages are prepared as described in U.S. Pat. Nos.5,378,825; 5,386,023; 5,489,677; 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Peptide nucleic acids (PNAs) are prepared in accordance with any of thevarious procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5. They may also be prepared in accordance with U.S.Pat. Nos. 5,539,082; 5,700,922, and 5,719,262, herein incorporated byreference.

Those skilled in the art will appreciate that numerous changes andmodifications may be made to the preferred embodiments of the inventionand that such changes and modifications may be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

It is intended that each of the patents, applications, printedpublications, and other published documents mentioned or referred to inthis specification be herein incorporated by reference in theirentirety.

What is claimed is:
 1. An enema formulation comprising a solution oremulsion formulated for rectal administration as an enema, wherein saidsolution or emulsion comprises an oligonucleotide and at least about0.63% hydroxypropyl methyl cellulose (HPMC), without added penetrationenhancer, wherein following the rectal administration of said solutionor emulsion to an individual the oligonucleotide is taken up by at leastone cell in the gastrointestinal tract, and wherein said enemaformulation comprising a solution or emulsion formulated for rectaladministration as an enema is not a solid suppository.
 2. Theformulation of claim 1 comprising from about 0.63% to about 1.5%hydroxypropyl methyl cellulose (HPMC).
 3. The formulation of claim 1wherein the oligonucleotide comprises SEQ ID NO:
 1. 4. The formulationof claim 3 wherein the oligonucleotide comprises ISIS
 2302. 5. Theformulation of claim 1 wherein the amount of said HPMC is about 1.5%. 6.A method for rectal delivery of an oligonucleotide to at least one cellin a gastrointestinal tract of an individual comprising: identifying anindividual in need of said oligonucleotide, and rectally administeringto said individual an enema formulation comprising a solution oremulsion formulated for rectal administration as an enema, wherein saidsolution or emulsion comprises an oligonucleotide and at least about0.63% hydroxypropyl methyl cellulose (HPMC), without added penetrationenhancer, wherein following the rectal administration of said solutionor emulsion to said individual the oligonucleotide is taken up by atleast one cell in the gastrointestinal tract, and wherein said enemaformulation comprising a solution or emulsion formulated for rectaladministration as an enema is not a solid suppository.
 7. The method ofclaim 6 comprising from about 0.63% to about 1.5% hydroxypropyl methylcellulose (HPMC).
 8. The method of claim 6 wherein the oligonucleotidecomprises SEQ ID NO:
 1. 9. The method of claim 8 wherein theoligonucleotide comprises ISIS
 2302. 10. The method of claim 9 furthercomprising analyzing colon tissue for delivery of the oligonucleotide toat least one cell.
 11. The method of claim 9 wherein the oligonucleotideis detectable in colon tissue at least three hours after rectaladministration of the oligonucleotide.
 12. The method of claim 6 whereinthe amount of said HPMC is about 1.5%.
 13. The method of claim 6 whereinthe oligonucleotide is present at a concentration of 5 mg/mL.